Biglycan is highly expressed in tumor stroma, associated with prognosis and angiogenesis-related genes in human breast cancer patients
We first evaluated the expression levels of biglycan in normal 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 compartment of human breast cancers (Fig. 1b). The relationship between 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 correlated with 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 is negatively associated with the survival of breast cancer patients. Biglycan has been reported to be involved in cell migration and tube formation of ECs.  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 been shown 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. Biglycan mRNA expression was positively correlated with both PECAM1 and ANGPT2 levels in human breast 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.
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 pads of 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. [24, 26] 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 biglycan-secreting TECs .
We then investigated biglycan expression in the mouse E0771 tumor model. As expected, Bgn KO mice showed markedly 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 , we analyzed biglycan concentrations in the plasma of mice. Biglycan concentration was highest in tumor-bearing WT mice compared to non-tumor WT mice and tumor-bearing Bgn KO mice (Fig. 2e). Biglycan and CD31 were co-localized by immunofluorescence staining using CD31 and biglycan antibodies (Fig. 2f). Furthermore, to confirm the expression of biglycan in TECs, we isolated TECs from E0771 tumors in WT mice and characterized them as described previously . 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 were highly 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 blood vessels were normalized in Bgn KO mice
To determine the contribution of biglycan to angiogenesis, we first analyzed CD31 + blood vessel density in 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 blood vessels were significantly decreased in Bgn KO mice as compared to the WT (Fig. 2h, i). Additionally, the percentage of α-SMA + pericyte-covered blood vessels was significantly higher in Bgn KO mice, which indicated that 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 blood flow 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 in lectin-positive (functional) vessels in E0771 tumors of Bgn KO mice as compared to those in the WT mice, implying that intratumoral blood perfusion was enhanced in Bgn KO mice (Fig. 2l, m). Tumor vascular normalization is also known to elicit enhanced tumor oxygenation ; thus, we analyzed tumor hypoxia by staining tumor tissues with a Glut1 antibody (Fig. 2n, o). Hypoxic area was decreased in tumors of Bgn KO mice compared to WT, which indicated that biglycan knockout increases the functionality of blood vessels in tumors via vascular normalization.
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 . BGN was positively correlated with ANGPT2 expression in human breast cancers (Fig. 1f). We next confirmed that Angpt2 mRNA 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 and non-treated cells (Fig. 3c). Furthermore, biglycan 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, but rather 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 , we confirmed that E0771 cells expressed TLR2 and TLR4 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 Bgn-TLR2 or -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 and ERK 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 and controls (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 indicated that 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, and functions in organization of the assembly of the extracellular matrix . Furthermore, as a DAMP, biglycan potentiates renal inflammation and fibrotic renal disorders.  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.
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 . Furthermore, a fibrotic tumor microenvironment can suppress the immune response to cancer.  Therefore, we analyzed the mRNA expression of several immune cell markers (Cd4, Cd8, Klrb1c, CD27, and Adgre1) within the E0771 tumor tissues from WT and Bgn KO mice (Fig. 4d-h). We found that only Cd8a mRNA expression was significantly increased in Bgn KO mice (Fig. 4h), potentially indicating an accumulation of CD8 + T cells. We confirmed this hypothesis by FACS analysis, which demonstrated that the percentage of CD45 + CD8 + T cells was higher in Bgn KO mice compared to WT mice (Fig. 4i, j).
Biglycan inactivation enhances drug delivery and the antitumor effect of paclitaxel
Normalization of the tumor microenvironment could potentially improve drug delivery and efficiency of chemotherapy . 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, the chemotherapeutic agent paclitaxel significantly suppressed E0771 tumor growth in Bgn KO mice (Fig. 5c). The number of lymph node metastases was decreased in paclitaxel treated Bgn KO mice (1/5, 20%) as compared to paclitaxel treated WT mice (2/5, 40%) (Fig. 5d). Paclitaxel treatment also increased the incidence of lung 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.