Cong et al. Breast Cancer Research(2021) 23:51
https://doi.org/10.1186/s13058-021-01423-w

RESEARCH ARTICLE

Open Access

Inhibition of stromal biglycan promotes
normalization of the tumor
microenvironment and enhances
chemotherapeutic efficacy
Li Cong1,2, Nako Maishi1,2, Dorcas A. Annan1,2, Marian F. Young3, Hirofumi Morimoto2, Masahiro Morimoto1,2,
Jin-Min Nam4, Yasuhiro Hida5 and Kyoko Hida1*

Abstract
Background: Biglycan is a proteoglycan found in the extracellular matrix. We have previously shown that biglycan
is secreted from tumor endothelial cells and induces tumor angiogenesis and metastasis. However, the function ofstroma biglycan in breast cancer is still unclear.
Methods: Biglycan gene analysis and its prognostic values in human breast cancers were based on TCGA data.
E0771 breast cancer cells were injected into WT and Bgn KO mice, respectively.
Results: Breast cancer patients with high biglycan expression had worse distant metastasis-free survival.
Furthermore, biglycan expression was higher in the tumor stromal compartment compared to the epithelial
compartment. Knockout of biglycan in the stroma (Bgn KO) in E0771 tumor-bearing mice inhibited metastasis tothe lung. Bgn KO also impaired tumor angiogenesis and normalized tumor vasculature by repressing tumor necrosis
factor-ɑ/angiopoietin 2 signaling. Moreover, fibrosis was suppressed and CD8+ T cell infiltration was increased intumor-bearing Bgn KO mice. Furthermore, chemotherapy drug delivery and efficacy were improved in vivo in Bgn
KO mice.
Conclusion: Our results suggest that targeting stromal biglycan may yield a potent and superior anticancer effectin breast cancer.
Keywords: Tumor stroma, Angiogenesis, Biglycan, Tumor microenvironment, Breast cancer

Background
Tumor vasculature is structurally abnormal relative tonormal vasculature and is characterized by highly permeable and leaky blood vessels [1]. The abnormality oftumor vasculature impairs blood perfusion and oxygenation, resulting in hypoxia and acidosis, and thus promoting tumor growth and progression. Furthermore, the* Correspondence: khida@den.hokudai.ac.jp
1
Vascular Biology and Molecular Pathology, Graduate School of Dental
Medicine, Hokkaido University, Sapporo 060-8586, Japan
Full list of author information is available at the end of the article

leakiness of tumor blood vessels results in spontaneous
hemorrhages and elevated interstitial fluid pressure (IFP)[2]. Hypoperfusion, hypoxia, and high IFP impede thefunctions of immune cells in tumors, in addition to
impeding the transport of therapeutic agents [3]. As aresult, abnormal tumor vasculature can promote drug
resistance and hinder anticancer activity.
Antiangiogenic therapy has been considered as apromising treatment strategy for breast cancer [4]. Bevacizumab, a neutralizing antibody against vascular endothelial growth factor (VEGF), mediates antitumor effects© The Author(s). 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License,
which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give
appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate ifchanges were made. The images or other third party material in this article are included in the article's Creative Commonslicence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain
permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.
The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to thedata made available in this article, unless otherwise stated in a credit line to the data.

Cong et al. Breast Cancer Research(2021) 23:51

by blocking tumor blood supply [5]. Furthermore, increased pericyte recruitment and vessel functions using
bevacizumab has been shown in breast cancer patients[6, 7]. Bevacizumab received FDA approval for metastatic breast cancer in 2008. However, other trials foundno significant effect on prolonging progression-free survival using Bevacizumab resulting in withdrawal of FDA
approval [8, 9]. Thus, alternative strategies to modulate
abnormal tumor vasculature in breast cancer are stillneeded.
Tumor endothelial cells (TECs) differ from normal
endothelial cells (NECs) in several aspects, such as gene
expression profiles [10], proangiogenic properties [11],sensitivity to drugs [12], and responses to growth factors[13]. Furthermore, TECs are cytogenetically abnormal[14]. By comparing the gene expression profiles of TECs
and NECs, we have previously shown that biglycan gene
expression levels are significantly elevated in isolated
TECs [15].
Biglycan is a small leucine-rich proteoglycan (SLRP),
consisting of a 42-kDa core protein and chondroitin sulfate/dermatan sulfate side chains [16]. Biglycan is involved in the migration of lung fibroblasts, theinflammatory response in renal macrophages, and abnormal collagen fibril morphology in bones [17–19].
Under physiological conditions, biglycan is sequesteredin the extracellular matrix (ECM). Biglycan is proteolytically cleaved from the ECM upon tissue stress or injury in a soluble form, thereby acting as a damageassociated molecular pattern (DAMP) protein [20].
Biglycan secreted from TECs can also induce proangiogenic effects in an autocrine manner [15] and has beenshown to promote tumor cell intravasation and metastasis through the NF-κB and ERK signaling pathways [21].
Biglycan has also been detected in several types of human cancers cells, such as endometrial cancer, bladdercancer, and pancreatic adenocarcinoma [22–24]. Functionally, biglycan expressed in cancer cells is involved intumor growth and progression, depending on tumor
type. However, the role of biglycan in tumor stroma has
not been clarified. In the present study, we focused on
the functional role of stromal biglycan in breast cancer
microenvironment using biglycan knockout (Bgn KO)
mice. Additionally, we investigated the therapeutic efficacy of targeting stromal biglycan combined with conventional chemotherapy in breast cancer.

Materials and methods
Bioinformatic analysis

We compared biglycan mRNA levels of human breast
cancers, normal mammary gland, and tumor stromaland epithelial parts using Oncomine (https://www.
oncomine.com/). The prognostic significances of biglycan mRNA and protein levels in human breast cancers

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were evaluated using the Kaplan-Meier plotter (http://
kmplot.com/analysis/). Co-expression of biglycan andangiogenesis-related genes was determined using the
cBioPortal database (https://www.cbioportal.org/).
Cell lines and culture conditions

MS1 cells (mouse immortalized islet-derived normal
endothelial cells) were obtained from American Type
Culture Collection (Manassas, VA). MS1 cells were cultured in Dulbecco’s minimum essential medium(DMEM, Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% heat-inactivated fetal bovine serum(FBS) and 1% penicillin/streptomycin antibiotics (SigmaAldrich). E0771 cells were purchased from CH3 BioSystems and cultured in RPMI 1640 medium (Sigma-Aldrich) supplemented with 10 mM HEPES and 10% FBS.
RAW264.7 cells were obtained from DS Pharma Biomedical and cultured in DMEM with 10% FBS. Dermal
endothelial cells from C57BL/6 mice were purchasedfrom Cell Biologics and cultured in EGM-2MV medium(Lonza, Basel, Switzerland). NIH3T3 cells from ATCC
were cultured in DMEM with 10% FBS. All cells were
cultured at 37 °C in a humidified atmosphere containing
5% CO2. The absence of Mycoplasma pulmonis was verified by polymerase chain reaction (PCR).
Mouse tumor xenograft model and drug administration

All animal experiments were approved by the animal research authorities of Hokkaido University. Wild type
(WT) C57BL/6 mice (female, 7–8 weeks old, 17–20 g)
were purchased from CLEA Japan (Tokyo, Japan). Bgn
KO C57BL/6 mice (female, 7–9 weeks old, 18–20 g) wereused in this study. Bgn KO C57BL/6 mice were established by Marian F. Young (National Institutes of Health,
USA) [25]. Briefly, Bgn-deficient mice were originallygenerated by using homologous recombination in embryonic stem cells. Biglycan was disrupted by inserting
the PGK-neo cassette from the pPNT vector into exon 2
at the Tth111I site. The genotype of Bgn KO mice wasdetermined by a PCR-based assay using the combinationof a primer containing the PGK promoter sequences(5′-tggatgtggaatgtgtgcgagg-3′) of the targeted allele
along with a forward primer corresponding to the 5′end of exon 2 (5′-caggaacattgaccatg-3′) and a reverse
primer corresponding to the 3′-end of exon 2 (5′-gaaaggacacatggcactgaag-3′). Biglycan was disrupted throughout the whole cells of the body. E0771 cells weretransfected with lentiviral vectors encoding the tdTomato and luciferase genes as previously described [21].
tdTomato/luc2-expressing E0771 cells (1 × 106 cells) suspended in an equal volume of Hank’s balanced salt solution (HBSS) and Matrigel (Corning, 356231) were
orthotopically implanted into the left fourth mammary
gland of WT and Bgn KO mice (5 mice per group).

Cong et al. Breast Cancer Research(2021) 23:51

Tumor volumes were measured with a caliper every 3 or
4 days and calculated using the formula: volume (V) =largest dimension × smallest dimension2 × 0.5. For histological analysis of tumors, mice were sacrificed 24 days
after implantation. Lungs were analyzed for tumor metastasis by ex vivo bioluminescence imaging using the
IVIS Spectrum system (Caliper Life Science). To evaluate the effect of Bgn KO on chemotherapy, mice wererandomly assigned to 4 groups (4 or 5 mice per group)
after tumor formation (7 days after tumor cell inoculation), and treated with paclitaxel (Taxol, EnZo,BML0T104) (2 mg/kg) or DMSO as a control via intraperitoneal (i.p.) injection every 3 days, for a total of 6 injections. After 22 days, mice were sacrificed via cervicaldislocation after isoflurane anesthesia. Tumor tissues,
lungs, and left inguinal lymph nodes were dissected. The
IVIS imaging system was used to detect metastasis inlungs and lymph nodes.
Isolation of TECs

TECs derived from E0771 tumors were isolated as previously described, with some modifications [14]. Briefly,
E0771 tumors grown in C57BL/6 mice (n = 10) wereminced. TECs were sorted using a magnetic cell sorting
system (Miltenyi Biotec, Tokyo, Japan) with anti-mouse
CD31 microbeads according to the manufacturer’s instructions. To improve the purity of TECs, CD45−/CD31+ cells were sorted using a FACS Aria II (BD
Biosciences). Isolated cells were maintained in EGM2MV medium (Lonza, Basel, Switzerland) with 15% FBS.
All purified ECs were cultured in EGM-2MV and wereused between passages 12 and 20.
Histological analysis

Tumor tissues were dissected from mice and processed
for embedding in paraffin or in OCT compound. Paraffin sections (4 μm) were stained with anti-mouse CD31
antibody (Abcam, ab28364) and counterstained withhematoxylin. Frozen tissue samples were cut into 10-μm
sections using a cryostat (Leica CM3050S, Leica Biosystems, Wetxlar, Germany). Sections were fixed in 100%
ice-cold acetone for 30 min and permeabilized with 0.1%
Triton in PBS at room temperature (RT) for 15 min.
Sections were then blocked in PBS containing 5% bovine
serum albumin (BSA) or 5% goat serum and 0.05%
Triton X-100. Frozen tumor tissues were double stainedusing Alexa Fluor 647-conjugated anti-mouse CD31
antibody (Biolegend, 102416) and anti-biglycan (Kerafast, LF-159) antibodies to determine the co-localizationof CD31 and biglycan in ECs. Pericyte-covered tumor
blood vessels in frozen sections were visualized by theco-staining of rat anti-mouse CD31 (BD Pharmingen,553370) and rabbit anti-mouse α-SMA (Abcam, ab5694)
followed by Alexa Fluor 647-conjugated goat anti-rat

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IgG (Biolegend, 405416) and Alexa Fluor 488-conjugatedgoat anti-rabbit IgG (Invitrogen, 11034) for 2 h at room
temperature. To detect hypoxia in tumors, frozen sections were stained with an anti-glucose transporter
Glut1 antibody (Abcam, ab115730) followed by Alexa
Fluor 647-conjugated goat anti-rabbit secondary antibody (Invitrogen, 21244). CD8+ T cells in frozen sections were stained by rat anti-mouse CD8a (Biolegend,
100702) followed by Alexa Fluor 647-conjugated goatanti-rat IgG (Biolegend, 405416). Frozen sections werecounterstained
with
4,6-diamidino-2-phenylindole(DAPI; Dojin), and images were obtained using a BZX810 microscope equipped with BZ-X800 Analyzer software (Keyence Corporation, Itasca, IL, USA). The areaof CD31+ or Glut1+ staining was quantified using ImageJ software (NIH), and the percentage of CD31+ orGlut1+ area was calculated as a percentage of the positive area to the total area. The area of pericyte-coveredtumor blood vessels was calculated by the ratio of thenumber of blood vessels co-stained with α-SMA and
CD31 to the total number of CD31-positive blood vessels. Collagen accumulation was evaluated by Picro Sirius Red Stain Kit (Abcam, ab150681). Five fields pertumor were quantified and values were averaged to obtain one value for each tumor. Each group consisted of 4
or 5 mice.
Assessment of perfused blood vessels

Doxorubicin (15 mg/kg, Sigma) was injected into the tailvein of E0771 tumor-bearing mice. FITC-lectin (2 mg/
kg, Vector Laboratories) was intravenously injected via
tail vein 4 h after doxorubicin injection. Tumors wereextracted 5 min after injection and frozen in blocks.
These frozen sections were stained with Alexa Fluor
488-conjugated anti-mouse CD31 antibody (Biolegend,
12514) or Alexa Fluor 647-conjugated anti-mouse CD31
antibody (Biolegend, 102416). The area of lectin- or
doxorubicin-positive staining was determined using
ImageJ software.
RNA isolation, reverse transcription PCR, and quantitative
real-time PCR

RNA from cells and tissues was isolated using the ReliaPrep RNA tissues miniprep system (Promega) and theRNeasy Micro kit (Qiagen), respectively. cDNA, reverse
transcription, PCR, and quantitative real-time PCR were
performed as previously described [26]. The primers
used in this study are listed in Supplementary Table S1.
ELISA

Biglycan concentration in mouse plasma was determinedusing an ELISA kit for mouse biglycan (Cloud-clone
Corp. SEJ226Mu). TNF-ɑ concentration in mouseplasma was determined using a Mouse TNF-ɑ

Cong et al. Breast Cancer Research(2021) 23:51

Quantikine ELISA kit (R&D systems, MTA00B) according to the manufacturer’s instructions. Absorbance was
detected using a microplate reader (Promega GloMax
Multi Detection System, Promega) at 450 nm.
Stimulation of cells with recombinant protein

E0771 cells were cultured in serum-free medium for 24
h before treatment with 5 μg/ml recombinant biglycan(Genway, GWB-ATG116) in serum-free RPMI medium.
After incubation for 6 h with recombinant biglycan protein, RNA was isolated. E0771 cells were pretreated with
anti-TLR2 (1 μg/ml, Biolegend, #246294) or TLR4
(10 μg/ml, Biolegend, #117608) antibody for 2 h. E0771
cells were then treated with serum-free RPMI media
containing 5 μg/ml biglycan for 6 h before RNA was extracted. E0771 cell were pretreated with NF-κB inhibitor
BAY11-7082 (10 μM, Calbiochem) or MEK inhibitor
U1026 (20 μM, CST) for 1 h. E0771 cells were thentreated with serum-free RPMI media containing 5 g/ml
biglycan for 6 h before RNA was extracted. MS1 cells
were kept in serum-free medium for 24 h before treatment with 1 g/ml TNF-ɑ (Peprotech, 300-01A) inserum-free DMEM medium. After incubation for 6 hwith recombinant biglycan protein, RNA was isolated.
NIH3T3 cells were cultured in serum-free DMEM for
24 h before treatment with 5 μg/ml recombinant biglycan in serum-free DMEM medium. After incubation for
6 h with recombinant biglycan protein, RNA was
isolated.
Biglycan knockdown using siRNA

Biglycan siRNA (5′-UAGAGGUGCUGGAGGCCUUUFAAGU-3′, 5′ -ACUUCAAAGGCCUCCAGCACCUCUA-3′)
was transfected into E0771-TECs using Lipofectamine
RNAi MAX Transfection Reagent (Invitrogen, Carlsbad,
CA, USA) according to the manufacturer’s instructions.
Control siRNA (Invitrogen, Carlsbad, CA, USA) wasused as a negative control. Knockdown efficiency wasconfirmed by quantitative real-time PCR.
Cell preparation and flow cytometry

E0771 cells were stained with PE-conjugated anti-mouse
CD284 (Biolegend, 117605) and Alexa Fluor 488conjugated anti-mouse CD282 (Biolegend, 121807). To
analyze CD8+ T cell infiltration in tumor tissues, E0771
tumors from WT and Bgn KO mice were dissected anddigested with collagenase II and DNase I. Single-cell suspensions were stained with FITC-conjugated anti-CD45
antibody (Biolegend, 103115) and APC-conjugated antiCD8a antibody (Biolegend, 100711). CD45 + CD8+ T
cells were analyzed using a FACS Aria II. Data was analyzed using FlowJo V10 software (Tree Star Inc.).

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Statistical analysis

All data are expressed as means ± standard deviation(SD) of three independent experiments performed intriplicate. Two-sided Student’s t test followed by Wilcoxon rank test was used for comparison between two
groups. Comparisons between multiple groups weremade using one-way ANOVA, followed by a TukeyKramer test. One-way ANOVA and T tests were analyzed using IBM SPSS software. p values < 0.05 wereconsidered significant.

Results
Biglycan is highly expressed in tumor stroma, associatedwith prognosis and angiogenesis-related genes in humanbreast cancer patients

We first evaluated the expression levels of biglycan innormal mammary glands and in human breast cancer
tissues using the Oncomine database (https://www.
oncomine.com/). Biglycan was found to be upregulated in human breast cancers compared to normal
mammary glands (Fig. 1a). Furthermore, biglycan expression was higher in the tumor stroma compartment compared to the tumor epithelial compartmentof human breast cancers (Fig. 1b). The relationshipbetween biglycan expression and prognosis of breast
cancer patients was investigated using Kaplan-Meier
Plotter database (http://kmplot.com/analysis/). Higher
mRNA and protein expression of biglycan correlatedwith worse distant metastasis-free survival of breast
cancer patients (Fig. 1c, d). These results suggested
that biglycan is indeed upregulated in breast cancer,
especially in tumor stroma, and its expression isnegatively associated with the survival of breast cancer patients. Biglycan has been reported to be involved in cell migration and tube formation of ECs[15]. To determine the effect of biglycan on angiogenesis in human cancers, we analyzed the correlation
between biglycan and angiogenesis-related gene expression in human breast cancers using the cBioPortal
database (https://www.cbioportal.org/). Platelet endothelial cell adhesion molecule (PECAM)-1 has beenshown to facilitate the interaction of endothelial cells
and play a role in angiogenesis. Angiopoietin 2(ANGPT2) destabilizes blood vessels by inducing detachment of pericytes from the endothelial cells. Pearson’s correlation coefficients showed that Biglycan
mRNA expression was positively correlated with both
PECAM1(Spearman’s correlation = 0.45) and ANGP
T2 (Spearman’s correlation = 0.4) levels in humanbreast cancers (Fig. 1e, f). Taken together, these results suggest that biglycan may be involved in tumor
angiogenesis and destabilization of tumor blood vessels
in human breast cancers.

Cong et al. Breast Cancer Research(2021) 23:51

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Fig. 1 Biglycan is highly expressed in tumor stroma, associated with angiogenesis gene expression and prognosis of human breast cancer
patients. a Comparison of biglycan mRNA expression was analyzed in normal mammary glands (n = 61) and breast cancer tissue (n = 389) using
Oncomine. b Comparison of biglycan mRNA expression was analyzed in 14 patient-matched tumor epithelium and tumor-associated stroma
specimens using Oncomine. c Kaplan-Meier analysis was used to assess breast cancer patients with high or low biglycan mRNA expression in
distant metastasis-free survival (DMFS) using a Kaplan-Meier plotter tool. d Kaplan-Meier analysis was used to assess breast cancer patients withhigh or low biglycan protein expression in DMFS using a Kaplan-Meier plotter tool. e Correlation between BGN and PECAM1 expression using
cBioPortal database. f Correlation between BGN and ANGPT2 expression using cBioPortal database

Cong et al. Breast Cancer Research(2021) 23:51

Lung metastasis was decreased in Bgn KO mice

To investigate how stromal biglycan affects tumor
growth in vivo, we orthotopically implanted murine
E0771 breast carcinoma cells into the mammary fat padsof WT and Bgn KO female mice. Bgn deficiency resulted
in no significant toxic effects in mice, except for a reduced growth rate and decreased bone mass [25, 27]. No
significant differences were seen in primary E0771 tumor
growth and tumor weight between the WT and Bgn KO(Fig. 2a, b). Different concentrations of recombinant
biglycan also had no significant effect on proliferation of
E0771 cells in vitro (Supplementary Fig. 1). However,
Bgn KO mice showed reduced lung metastasis by IVIS
(Fig. 2c). As E0771 cells had low expression of biglycan
in vitro (Supplementary Fig. 2a), these results suggested
that stroma biglycan is involved in lung metastasis but
not in tumor growth, consistent with our previous report showing induction of metastasis by biglycansecreting TECs [21].
We then investigated biglycan expression in the mouse
E0771 tumor model. As expected, Bgn KO mice showedmarkedly reduced levels of biglycan mRNA expression
in tumor tissues compared to WT mice (Fig. 2d). As
biglycan is secreted in a soluble form during inflammation [17], we analyzed biglycan concentrations in theplasma of mice. Biglycan concentration was highest intumor-bearing WT mice compared to non-tumor WT
mice and tumor-bearing Bgn KO mice (Fig. 2e). Biglycan
and CD31 were co-localized by immunofluorescencestaining using CD31 and biglycan antibodies (Fig. 2f).
Furthermore, to confirm the expression of biglycan in
TECs, we isolated TECs from E0771 tumors in WT miceand characterized them as described previously [15].
TECs were shown to be negative for the monocyte
markers CD11b and CD45, and positive for EC markers
CD31, CD144, and CD105 by RT-PCR (Supplementary
Fig. 2b). TECs were positive for EC markers CD31,
CD105, and lectin, and negative for the hematopoietic
marker CD45 by flow cytometry analysis (Supplementary
Fig. 2c). These results indicate that isolated cells werehighly purified ECs. We analyzed Bgn expression in
TECs isolated from E0771 tumors (E0771-TECs), isolated CD31-negative cells including tumor cells, fibroblasts, and immune cells, and commercially available
dermal ECs from normal mice (NECs) by real-time PCR.
Bgn mRNA expression was significantly higher in
E0771-TECs compared to CD31-negative cells and dermal ECs (Fig. 2g), indicating that TECs were a source of
biglycan in E0771 tumor.
Tumor angiogenesis was impaired and tumor bloodvessels were normalized in Bgn KO mice

To determine the contribution of biglycan to angiogenesis, we first analyzed CD31+ blood vessel density inPage 6 of 17

normal mammary glands from WT and Bgn KO mice.
No significant changes were observed in morphology
and density of normal mammary gland blood vessels in
Bgn KO and WT mice (Supplementary Fig. 3a, b). Next,
we evaluated the microvessel density in E0771 tumors
from WT and Bgn KO mice. The CD31+ tumor bloodvessels were significantly decreased in Bgn KO mice ascompared to the WT (Fig. 2h, i). Additionally, the percentage of α-SMA+ pericyte-covered blood vessels was
significantly higher in Bgn KO mice, which indicatedthat more mature blood vessels existed in tumors from
Bgn KO mice compared to WT mice (Fig. 2j, k). These
data suggested that tumor blood vessels in Bgn KO mice
are structurally normalized.
Since blood vessel normalization can also enhance bloodflow within vessels, we further assessed the vascular function in E0771 tumors in WT and Bgn KO mice. First, by
intravenous lectin injection, we identified an increase inlectin-positive (functional) vessels in E0771 tumors of Bgn
KO mice as compared to those in the WT mice, implyingthat intratumoral blood perfusion was enhanced in Bgn
KO mice (Fig. 2l, m). Tumor vascular normalization is
also known to elicit enhanced tumor oxygenation [3];thus, we analyzed tumor hypoxia by staining tumor tissues
with a glucose transporter (Glut1) antibody (Fig. 2n, o).
Glut1+ area was decreased in tumors of Bgn KO micecompared to WT mice. As hypoxic response is mainly ascribed to hypoxia-inducible factor-1 (HIF-1) α which is involved in the induction of Glut1 expression [28], we
checked whether biglycan affects HIF1-α and Glut1 expression in tumor cells. We found that Hif1a and Slc2a1
expression elevation in E0771 cells by stimulation with recombinant biglycan (Supplementary Fig. 4 a, b), which indicated that biglycan might mediate HIF1-α and Glut1
expression in tumor cells. Together, these data suggested
that alleviating tumor hypoxia by biglycan knockout might
be due to enhancement of vascular normalization and mediation of HIF1-α and Glut1 expression in tumor cells.
Angpt2 expression was decreased in E0771 tumors from
Bgn KO mice

Vascular normalization is regulated by several molecules,
such as ANG-TIE, PDGFB, and NG2 [29]. BGN was
positively correlated with ANGPT2 expression in humanbreast cancers (Fig. 1f). We next confirmed that Angpt2mRNA expression was decreased in tumor tissues from
Bgn KO mice (Fig. 3a). To determine whether biglycan
has any direct effect on Angpt2 expression, we stimulated MS1 cells (immortalized normal ECs) with recombinant biglycan after confirming the expression of the
biglycan receptors, Toll-like receptors (TLRs) 2 and 4 in
the MS1 cells (Fig. 3b). There was no significant difference in Angpt2 expression between biglycan-treated andnon-treated cells (Fig. 3c). Furthermore, biglycan

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Fig. 2 Stromal biglycan inhibition suppresses lung metastasis, by normalizing tumor vasculature in breast cancer. a Growth curves of E0771
tumor growth in WT and Bgn KO mice measured every 4 days. b Comparison of E0771 tumor weight at day 24 after inoculation. c Representative
IVIS images and quantitative analysis of metastatic lungs in E0771 tumors of WT versus Bgn KO mice. d Bgn mRNA expression in E0771 tumor
tissues of WT and Bgn KO mic analyzed by quantitative real-time RT-PCR. e Biglycan concentration in mouse plasma from non-tumor WT mice,
E0771 tumor-bearing WT mice, and E0771 tumor-bearing Bgn KO mice detected by ELISA. f Immunofluorescent images of biglycan (green)
expression in CD31+ blood vessels (red) in E0771 tumors grown in WT mice. DAPI, blue. Scale bar = 20 μm. g Bgn mRNA expression in FACS
isolated CD31− cells, CD45− CD31+ cells (E0771-TECs), and dermal ECs from naïve mice by quantitative real-time RT-PCR. h Representative
images of CD31+ blood vessels in E0771 tumors from WT and Bgn KO mice. Scale bar = 100 μm. i Quantification of blood vessel density. j
Representative images of CD31 immunostaining (red), ɑ-SMA (green), and DAPI nuclear staining (blue) in E0771 tumors from WT and Bgn KO
mice. Scale bar = 100 μm. k Quantification of ɑ-SMA+ CD31+ tumor blood vessels. The rate of microvessel pericyte coverage was analyzed bycounting the vessels that stained positive for both CD31 and α-SMA (white arrowheads) among CD31-positive vessels. l Representative images of
CD31 immunostaining (red), FITC-lectin (green), and DAPI (blue) in E0771 tumors from WT and Bgn KO mice. Scale bar = 100 μm. m Quantificationof lectin+ blood vessels. The rate of lectin+ blood vessels was analyzed by counting the vessels that stained positive for both CD31 and lectin(white arrowheads) among CD31-positive vessels. n Representative images of CD31 immunostaining (green), Glut1 (red), and DAPI (blue) in E0771
tumors from WT and Bgn KO mice. Scale bar = 100 μm. o Quantification of Glut1+ area. *p < 0.05, ***p < 0.001. Significance in b, d, i, k, m, ando were determined by a two-tailed Student’s t test. Significance in g was determined by a one-way ANOVA. All data represent means ± SD

knockdown in E0771-TECs (Fig. 3d) did not alter
Angpt2 mRNA expression in the cells. (Fig. 3e). Therefore, we speculated that biglycan may not regulate
Angpt2 expression in ECs directly via its receptors, butrather by other indirect mechanisms. We further analyzed the molecular relationship between biglycan and
ANGPT2 by Ingenuity Pathway Analysis (IPA, Tomy
Digital Biology), and found that TNF was known to mediate this interaction (Fig. 3f). We therefore hypothesized that ANGPT2 might be regulated by biglycan/
TNF-α signaling, leading to the destabilization of tumor
blood vessels.
TNF-ɑ-enhanced Angpt2 expression is controlled by
biglycan through activation of NF-κB and ERK via TLR2/4

We compared Tnf mRNA expression and TNF-ɑ secretion in E0771 tumor-bearing WT and Bgn KO mice. Bgn
KO mice showed significantly reduced Tnf mRNA expression in E0771 breast cancer tissues and TNF-ɑ secretion in plasma from tumor-bearing mice (Fig. 3g, h).
As soluble biglycan can bind to TLRs 2 and 4 on macrophages and activate mitogen-activated protein kinase(MAPK) p38, extracellular signal-regulated kinase (ERK),
and nuclear factor-κB (NF-κB) signaling pathways [30],we confirmed that E0771 cells expressed TLR2 andTLR4 by flow cytometry analysis (Fig. 3i). We proceeded
to investigate the paracrine effects of biglycan on TNF-ɑ
expression in E0771 cells (Fig. 3j) and RAW macrophages (Supplementary Fig. 4a) stimulated with recombinant biglycan. Tnf mRNA expression was significantly
increased in biglycan-stimulated cells as compared to
non-treated control cells (Fig. 3j). Blockage of the BgnTLR2 or Bgn-TLR4 interaction in E0771 cells by neutralizing antibodies significantly decreased Tnf mRNA
expression in E0771 cells stimulated with recombinant
biglycan (Fig. 3k, l), suggesting that biglycan regulates
TNF-ɑ expression in tumor cells through binding to
TLR2 and TLR4. To elucidate the downstream

mediators of Tnf induction, E0771 cells were first pretreated with the NF-κB inhibitor BAY11-7082 or the
ERK inhibitor U1026, and then stimulated with recombinant biglycan. NF-κB or ERK blockade decreased Tnf
mRNA expression (Fig. 3m, n), indicating that biglycan
may regulate TNF-ɑ expression through NF-κB andERK signaling pathways.
Furthermore, we analyzed the effect of biglycan on
TNF-ɑ expression in ECs. The Tnf mRNA expression
level in MS1 cells was not changed significantly after
stimulation with recombinant biglycan (Supplementary
Fig. 4b). In addition, Tnf expression showed no difference between biglycan knockdown E0771-TECs andcontrols (Supplementary Fig. 4c), implying that biglycan
has no autocrine effect on TNF-ɑ expression. In order
to confirm that TNF-α can indeed induce Angpt2 expression in ECs, we confirmed the presence of TNFR1
and TNFR2 expression in ECs (Supplementary Fig. 5)
and stimulated MS1 cells with TNF-α. This led to increased levels of Angpt2 mRNA in MS1 cells (Fig. 3o),consistent with previous reports. These data indicatedthat biglycan may regulate Angpt2 expression via enhanced levels of TNF-α indirectly in a paracrine manner.
Biglycan inhibition suppresses tumor fibrosis

Biglycan has been shown to interact with collagen I andfunctions in organization of the assembly of the extracellular matrix [31]. Furthermore, as a DAMP, biglycan potentiates renal inflammation and fibrotic renal disorders[32]. Thus, we assessed collagen deposition by picrosirius red staining in E0771 tumors. We found that collagen accumulation was reduced in tumors from Bgn KO
mice compared to those in WT mice (Fig. 4a, b). Col1a1
mRNA expression was also decreased in tumor tissues
from Bgn KO mice (Fig. 4c). These findings indicate that
biglycan knockout suppressed tumor fibrosis. As activated cancer-associated fibroblasts (CAFs) in tumor
stroma is involved in tumor fibrosis and activated CAFs

Cong et al. Breast Cancer Research

Fig. 3 (See legend on next page.)(2021) 23:51

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Cong et al. Breast Cancer Research(2021) 23:51

Page 10 of 17(See figure on previous page.)
Fig. 3 TNF-ɑ-enhanced Angpt2 expression is controlled by biglycan in E0771 tumors. a Angpt2 mRNA expression in E0771 tumor tissues of WT
and Bgn KO mice by quantitative real-time PCR. b TLR2 and TLR4 expression in MS1 cells analyzed by FACS analysis. Blue: antibody. Black: isotype.
c Angpt2 mRNA expression in MS1 cells treated with recombinant biglycan (5 g/ml) by quantitative real-time RT-PCR. d Bgn mRNA expression in
E0771-TECs transfected with Bgn siRNA by quantitative real-time PCR. e Angpt2 mRNA expression in E0771-TECs transfected with Bgn siRNA by
quantitative real-time PCR. f Flowchart of biglycan, TNF-ɑ, and Angpt2 signaling by IPA analysis. g Tnf mRNA expression by quantitative RT-PCR in
E0771 tumor tissues from WT and Bgn KO mice. h TNF-ɑ secretion in the plasma of E0771 tumor-bearing mice was detected by ELISA. Each dot
represents one mouse. i TLR2 and TLR4 expression in E0771 cells analyzed by flow cytometry. Blue: isotype. Black: antibody. j Tnf mRNA
expression in E0771 cells treated with recombinant biglycan (5 μg/ml). k, l After blocking of TLR2 or (k) TLR4 (l), E0771 cells were stimulated byrecombinant biglycan (5 μg/ml). Tnf mRNA expression was analyzed by quantitative RT-PCR. m, n After treating with (m) the NF-κB inhibitor(10 μM) or (n) the MEK inhibitor (20 μM), E0771 cells were stimulated by recombinant biglycan (5 μg/ml). Tnf mRNA expression was analyzed byquantitative real-time RT-PCR. o Angpt2 mRNA expression in MS1 cells treated with recombinant TNF-ɑ (10 ng/ml) by quantitative RT-PCR. *p <0.05, **p < 0.01, ***p < 0.001. NS, not significant. Significance was determined in a, c, g, j, and o by two-tailed Student’s t test, and in d, e, k, l, m,and n by a one-way ANOVA. All data represent means ± SD

can be identified by their expression of α-SMA [33], αSMA+ fibroblast (excluded pericytes) assessment bystaining was analyzed in biglycan KO mice. α-SMA+ fibroblasts were significantly decreased in Bgn KO mice ascompared to the WT mice (Fig. 4d, e). We also analyzed
the effect of biglycan on α-SMA expression in fibroblasts
and found that α-SMA mRNA expression was upregulated in NIH3T3 cells by biglycan treatment (Fig. 4f).
These results indicate that biglycan may activate fibroblasts via upregulating α-SMA expression.
Stromal biglycan deficiency increases the recruitment of
CD8+ T cells in breast cancer

The vascular normalization in tumor stroma is reported
to increase the accessibility of immune cells to the tumors [34]. Furthermore, a fibrotic tumor microenvironment can suppress the immune response to cancer [35].
Therefore, we analyzed the mRNA expression of several
immune cell markers (Cd4, Cd8, Klrb1c, CD27, andAdgre1) within the E0771 tumor tissues from WT and
Bgn KO mice (Fig. 4g–k). We found that only Cd8amRNA expression was significantly increased in Bgn KO
mice (Fig. 4k), potentially indicating an accumulation of
CD8+ T cells. We confirmed this hypothesis by FACS
analysis, which demonstrated that the percentage ofCD45 + CD8+ T cells was higher in Bgn KO mice compared to WT mice (Fig. 4l, m). The infiltration of CD8+
T cells in E0771 tumors was also analyzed by IHC.
Quantification of CD8+ T cells by IHC showed a significant increase in Bgn KO mice, with lymphocytes distributed throughout the tumor (Fig. 4n–p). Together, thesedata indicate that CD8+ T cell distribution in tumor
tissues may be improved by Bgn inhibition.
Biglycan inactivation enhances drug delivery and theantitumor effect of paclitaxel

Normalization of the tumor microenvironment could
potentially improve drug delivery and efficiency ofchemotherapy [36]. As tumor blood perfusion was improved and tumor fibrosis was inhibited in Bgn KO

mice, we measured drug delivery and chemotherapeutic
efficacy in E0771 tumors. Doxorubicin delivery was enhanced in Bgn KO mice (Fig. 5a, b). Additionally, thechemotherapeutic agent paclitaxel significantly suppressed E0771 tumor growth in Bgn KO mice (Fig. 5c).
The number of lymph node metastases was decreased inpaclitaxel-treated Bgn KO mice (1/5, 20%) as comparedto paclitaxel-treated WT mice (2/5, 40%) (Fig. 5d).
Paclitaxel treatment also increased the incidence oflung metastasis (2/5, 40%) in WT mice compared to
Bgn KO mice (0/5) (Fig. 5e). These data suggested
that loss of stromal biglycan enhanced chemotherapeutic
efficacy in tumors via normalization of breast cancer
microenvironment.

Discussion
Recently, the role of biglycan has been investigated incancer cells. Biglycan has bidirectional roles modulatingtumor growth and progression in different tumor types[37–39]. However, the roles of biglycan in breast cancer
microenvironment remain unclear. In the current study,
we found that stromal biglycan inhibition enhanced chemotherapeutic efficacy through normalization of not
only the vascular but also the tumor microenvironment,
resulting in increased oxygen perfusion and drug delivery (Fig. 6). To our knowledge, this is the first reportdemonstrating that stromal biglycan mediates
destabilization of the tumor microenvironment, suggesting that biglycan can potentially serve as a therapeutictarget in combination cancer therapies.
Tumor vascular normalization has been shown to enhance tumor oxygenation, reduce cancer progression,and decrease interstitial pressure in the tumor, whichconsequently enhances drug delivery [40]. Treatment
with bevacizumab prunes some abnormal vessels and remodels the remaining vessels during the normalization
window [41]. However, intrinsic and evasive drug resistance still develops, and the evaluation of thenormalization window is still a challenge. There are no
validated biomarkers for antiangiogenic agents toCong et al. Breast Cancer Research(2021) 23:51

Page 11 of 17

Fig. 4 Biglycan inhibition reduces stromal fibrosis and normalizes immune suppressive microenvironment. a Representative images of picrosirius
red staining for collagen in E0771 tumors from WT and Bgn KO mice. Scale bar = 100 μm. b Quantification of collagen accumulation. Data
represent means ± SD. n = 5 fields, 5 mice per group. c Col1a1 mRNA expression in E0771 tumor tissues of WT and Bgn KO mice by quantitative
RT-PCR. n = 4 RT-PCR replicates per mouse, 5 mice per group. d Representative images of α-SMA (green), CD31 immunostaining (red), and DAPI
nuclear staining (blue) in E0771 tumors from WT and Bgn KO mice. Scale bar = 100 μm.e Quantification of α-SMA + fibroblasts. The area of αSMA + fibroblasts was analyzed by excluding α-SMA + pericytes from CD31+ blood vessels. f α-SMA mRNA expression in NIH3T3 cells treated withrecombinant biglycan by quantitative real-time RT-PCR. g Cd4, h Klrb1c, i Cd27, j Adgre1, and k Cd8 mRNA expression by quantitative RT-PCR in
E0771 tumor tissues from WT and Bgn KO mice. NS, not significant. n = 4 RT-PCR replicates per mouse, 4–5 mice per group. l, m CD8 + CD45+ T
cell analysis in E0771 tumors of WT and Bgn KO mice by flow cytometry. l Representative data and m the percentage of CD8 + CD45+ T cells/
CD45+ T cells. n = 5 mice per group. n, o Representative images of CD8+ T cells in E0771 tumors from WT and Bgn KO mice. o in tumor center,
Scale bar = 100 μm. p Quantification of CD8+ T cells in tumor center. *p < 0.05, **p < 0.01, ***p < 0.001. Significance was determined by two-tailed
Student’s t testCong et al. Breast Cancer Research(2021) 23:51

Page 12 of 17

Fig. 5 Biglycan deletion enhances drug delivery and antitumor effect of paclitaxel in breast cancer. a Representative images of CD31 immunostaining(green), doxorubicin (red), and DAPI nuclear staining (blue) in E0771 tumors from WT and Bgn KO mice. Scale bar = 100 μm. b Quantification of drugdelivery. Significance was determined by two-tailed Student’s t test. n = 5 fields per mouse, 5 mice per group. c Growth curves of E0771 tumors grown
in WT and Bgn KO mice. Mice were treated with DMSO or 2 mg/kg paclitaxel once every 3 days for a total of 6 treatments. Significancewas determined by a one-way ANOVA. n = 4–5 mice per group. d, e Representative IVIS images of d metastatic lymph nodes and e lungs in E0771
tumors of DMSO- or paclitaxel-treated WT and Bgn KO mice as described in c. All data represent means ± SD. *p < 0.05, ***p < 0.001

maintain normalization of tumor vasculature, and propermolecular diagnostic markers remain to be discovered[40]. In the current study, high expression of stromal
biglycan associated with a worse outcome in humanbreast cancers, indicating that tumor stromal biglycan
might play a role in cancer progression.

Biglycan has been reported to be expressed in thetumor microenvironment [15, 21, 42]. We previously
showed that biglycan is highly expressed in TECs and facilitates cancer metastasis [15, 21]. In our study, biglycan
was only expressed in tumor blood vessels of mousebreast cancers, and not in normal mammary glandCong et al. Breast Cancer Research(2021) 23:51

Page 13 of 17

Fig. 6 Schematic illustrating mechanisms of biglycan modulating tumor microenvironment. Inactivation of tumor stromal biglycan normalizes
tumor vasculature via downregulation of Angpt2. Biglycan knockout in tumor stroma decreases tumor stiffness and increases the accumulation of
CD8+ T cells. Thus, targeting stromal biglycan can facilitate drug delivery in tumors

tissue. Furthermore, co-expression analysis by cBioPortal
showed that BGN was positively associated with PECA
M1 and ANGPT2 expression in human breast cancers.
Both PECAM1 and ANGPT2 genes are encoding theangiogenesis-related molecules, which means that BGN
is involved in regulating angiogenesis. Also, since
biglycan-deficient mice exhibited no embryonic lethality[25], targeting biglycan may not cause severe damage innormal tissue and is perhaps unlikely to cause adverse
effects. These data indicated that stromal biglycan may
be a clinically relevant therapeutic target in breast cancer.
We observed that biglycan-deficient tumor vessels
showed normalized vasculature phenotypes, such as increased pericyte coverage and decreased vessel perfusion, leading to a less hypoxic environment andincreased drug delivery. Also, biglycan depletion instroma suppressed tumor fibrosis, consistent with a previous report [43]. Normalization of the tumor microenvironment has also been theorized to enhance drugdelivery and the efficiency of chemotherapies [36]. Together, these results provide a rationale for the combination therapy of stromal biglycan blockade and theconventional chemotherapeutic agent, paclitaxel. Paclitaxel, widely used to treat breast cancer, both kills andactivates tumor cells, thereby increasing chemoresistanceand metastasis, which markedly limits its clinical application. Here we observed that paclitaxel had asignificantly greater effect on tumor growth in biglycan
knockout mice compared to WT mice. Therefore, targeting stromal biglycan may enhance the therapeuticpotential of chemotherapeutic agents in treating cancers.
We then investigated how inactivation of stromal
biglycan induced vascular normalization mechanistically.
Pericytes embedded in the basement membrane of bloodvessels are important regulators of angiogenesis and vascular stability [44]. Pericytes also promote maturation ofblood vessels by stabilizing endothelial cells [45]. ANGP
T2, which is largely secreted from activated ECs, is directly angiogenic, and binding of ANGPT2 to its receptor
TIE2 on endothelial cells destabilizes vessels by leading
to detachment of pericytes, thereby promoting vascularleakiness [46]. Targeting ANGPT2 signaling in tumor
vasculature can restore vascular stability and decrease
tumor growth and metastasis [47]. It has been shownthat ANGPT2 is downstream of biglycan signaling intelomerase-immortalized microvascular endothelial cells[48]. Furthermore, BGN was positively associated with
ANGPT2 expression in human breast cancers. We also
demonstrated that Angpt2 mRNA is downregulated instromal biglycan-deficient tumors. Our results suggestthat biglycan destabilizes tumor blood vessels by mediating Angpt2 expression. However, biglycan itself indirectly induced Angpt 2 expression in endothelial cells;
we propose that TNF-α stimulates tumor angiogenesis

Cong et al. Breast Cancer Research(2021) 23:51

via suppression of vascular integrity and increased perfusion as a result of biglycan signaling. TNF-α is a major
factor in inflammation and cancer progression [49].
Furthermore, TNF-α promotes angiogenesis in vivo [50]and remodels blood vessels during inflammation [51].
Targeting TNF-α increases mature blood vessels in
rheumatoid arthritis synovium [52].
Biglycan acts as a DAMP, and here we provided evidence demonstrating that stromal biglycan promotes
TNF-α expression in tumor cells through binding to
TLR2 or TLR4, and subsequent activation of NF-κB andERK signaling pathways. Consistent with previous reports [53], we also found that increased TNF-α expression led to upregulated Angpt2 mRNA in ECs. These
results indicated that stromal biglycan enhances TNF-α
expression in tumor cells and that TNF-α subsequently
upregulates Angpt2 expression in ECs, thus destabilizing
tumor blood vessels. Furthermore, expression of Mcr1, aM2-like macrophage-associated gene, was decreased intumor-bearing Bgn KO mice (data not shown), whichsuggests that M2-like macrophages may be altered bystromal biglycan deficiency. M2-like macrophages play a
critical role in promoting the formation of an abnormal
tumor vascular network and subsequent tumor progression and invasion [54]. Targeting of M2-like macrophages may also potentially lead to the normalization oftumor vasculature [55]. Biglycan has been demonstrated
to trigger the synthesis of the macrophage chemoattractants chemokine (C-C motif) ligand CCL2 and CCL5[56]; thus, it may be possible that biglycan deficiency
modulates tumor vasculature through recruitment ofmacrophages.
As a DAMP, biglycan induces inflammation and fibrogenesis [57]. Biglycan promotes clustering of TLR2/
TLR4 with the purinergic P2X7 and P2X4 receptors,
which activate NLRP3 [58]. The NLRP3 inflammasome
triggers secretion of IL-1β and IL-18, which are involved
in renal fibrogenesis [57, 59]. We also observed that
biglycan depletion in stroma suppressed tumor fibrosis
as well as downregulated collagen I expression. Furthermore, α-SMA + fibroblasts were fewer in biglycan depleted tumors. Biglycan may activate CAFs via
upregulating α-SMA expression, thus enhancing fibrosis.
Increasing ECM stiffness can enhance cancer progression and metastasis. Biglycan in fibroblasts promotes
melanoma invasiveness via increased tissue stiffness,thereby inducing integrin-β1 expression [43], which is
supported by our findings. The ECM in tumors can also
impair the function of blood vessels via compression[60]. Targeting stromal biglycan reduced desmoplasia
and overcame vascular compression. Furthermore, destabilizing tumor vasculature and increasing fibrosis canlead to tumor hypoxia [61]. We have found that biglycan
increased HIF1-α and Glut1 expression in tumor cells. It

Page 14 of 17

has been reported that biglycan increased the interaction
between NF-kB and the HIF-1a promoter, leading to enhanced promoter activity and increased HIF-1a mRNAlevels in endothelial cells [39], which is consistent with
our findings. HIF1-α is involved in the induction of
Glut1 expression [28]. These data indicated that biglycan
might be involved in tumor hypoxia response in tumor
cells through upregulating HIF1-α and Glut1 expression.
Alleviating hypoxia is highly beneficial for improving
cancer treatment [61]. However, the detailed mechanisms by which biglycan mediates tumor desmoplasia
and hypoxia remain unclear.
Triple negative breast cancer (TNBC) constitutes 15%of all breast cancers and is defined by the lack the expression of hormone receptors and HER2 [62]. Recently,
TNBC has been approved to be treated with an immune
checkpoint inhibitor (ICI) by the Food and Drug Administration [63]. Here, we provide evidence showing thattargeting stromal biglycan alters the tumor microenvironment and enhances intratumoral infiltration of CD8+
T cells in a TNBC mouse model (E0771 breast cancer).
However, further analysis is required to elucidate detailed mechanisms of the antitumor immune responses
following targeting of biglycan. Furthermore, as biglycan
activates the NF-κB signaling pathway, which is one
transcriptional activator of PD-L1 [64], we speculate that
biglycan may induce PD-L1 expression in tumor cells.
Thus, targeting biglycan may achieve superior antitumor
effects via multiple parallel pathways.

Conclusions
Our findings demonstrated that stromal biglycan alters
breast cancer microenvironment, and deletion of stromal
biglycan enhances the efficacy of chemotherapy treatment. Biglycan may represent a promising candidate topotentiate the efficacy of anticancer therapies in breast
cancer.
Supplementary Information
Supplementary information accompanies this paper at https://doi.org/10.
1186/s13058-021-01423-w.
Additional file 1:. Supplementary Figure.
Additional file 2:. Supplementary Table.

Acknowledgements
We would like to thank Dr. A. Matsuda, Mr. S. Baba, Ms. M. Sasaki, and Ms. Y.
Suzuki for their technical assistance with the experiments.
Authors’ contributions
L.C. performed and analyzed experiments and wrote the original manuscript.
N.M. developed the methodology and modified the manuscript. D.AA., H.M.,
and M.M. performed the experiments and analyzed data. M.F. and JM.N.
coordinated the research project. Y.H. helped conceive the study. K.H.
conceived, designed, and supervised the study and modified the manuscript.
All authors read and approved the final manuscript.

Cong et al. Breast Cancer Research(2021) 23:51

Funding
This research was supported by JSPS Grants-in-Aid for Scientific Research to
NM (JP18K09715), YH (JP18H02891), and KH (JP18H02996), Grants from Japan
Agency for Medical Research and Development (AMED) to NM(JP18ck0106198h0003) and KH (JP19ck0106406h0002), the Intramural Research Program of the NIH, NIDCR Molecular Biology of Bones and Teeth
Section (Z01DE000379-35) and the Veterinary Resources Core (ZIC DE00074005) to MY.

Page 15 of 178.

9.

Availability of data and materials
The datasets used during the current study are available from thecorresponding author on reasonable request.

10.

Declarations

11.

Ethics approval and consent to participate
All animal experiments were approved by the animal research authorities of
Hokkaido University. The authors followed the Animal Research: Reporting of
In Vivo Experiments (ARRIVE) guidelines for animal studies.

12.

Consent for publication
Not applicable.
13.
Competing interests
We declare no potential conflicts of interest.
Author details
1
Vascular Biology and Molecular Pathology, Graduate School of Dental
Medicine, Hokkaido University, Sapporo 060-8586, Japan. 2Vascular Biology,
Frontier Research Unit, Institute for Genetic Medicine, Hokkaido University,
Sapporo 060-0815, Japan. 3Molecular Biology of Bones and Teeth Section,
NIDCR, Bethesda, MD 20892-4320, USA. 4Global Institution for Collaborative
Research and Education (GI-CoRE), Faculty of Medicine, Hokkaido University,
Sapporo 060-0808, Japan. 5Department of Cardiovascular and Thoracic
Surgery, Faculty of Medicine, Hokkaido University, Sapporo 060-8638, Japan.

14.

15.

16.

Received: 6 January 2021 Accepted: 25 March 2021
17.
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