Targeting stromal cell Syndecan‐2 reduces breast tumour growth, metastasis and limits immune evasion

Abstract Tumour stromal cells support tumourigenesis. We report that Syndecan‐2 (SDC2) is expressed on a nonepithelial, nonhaematopoietic, nonendothelial stromal cell population within breast cancer tissue. In vitro, syndecan‐2 modulated TGFβ signalling (SMAD7, PAI‐1), migration and immunosuppression of patient‐derived tumour‐associated stromal cells (TASCs). In an orthotopic immunocompromised breast cancer model, overexpression of syndecan‐2 in TASCs significantly enhanced TGFβ signalling (SMAD7, PAI‐1), tumour growth and metastasis, whereas reducing levels of SDC2 in TASCs attenuated TGFβ signalling (SMAD7, PAI‐1, CXCR4), tumour growth and metastasis. To explore the potential for therapeutic application, a syndecan‐2‐peptide was generated that inhibited the migratory and immunosuppressive properties of TASCs in association with reduced expression of TGFβ‐regulated immunosuppressive genes, such as CXCR4 and PD‐L1. Moreover, using an orthotopic syngeneic breast cancer model, overexpression of syndecan‐2‐peptide in TASCs reduced tumour growth and immunosuppression within the TME. These data provide evidence that targeting stromal syndecan‐2 within the TME inhibits tumour growth and metastasis due to decreased TGFβ signalling and increased immune control.

of heterogeneous cell types including; mesenchymal stromal cells (MSCs), cancer associated fibroblasts (CAFs), immune cells and endothelium. 3 The importance of these cell types is indicated by the fact that a stroma-related gene signature predicts resistance to chemotherapy in breast cancer. 4 Therapies directed to cancer cells fail to eradicate stromal cells, which can re-establish a tumourigenic environment and promote recurrence. 5,6 Studies indicate that fibroblast activation protein alpha (FAP) + , fibroblast surface protein (FSP) + , alpha smooth muscle actin (αSMA) + , CD45 − and CD11b − stromal cells within lung and breast tumours promote tumourigenesis, creating an immunosuppressive niche within the tumour microenvironment (TME). [7][8][9] An FAP + , platelet-derived growth factor receptor (PDGFR)α + , PDGFRβ + , CD45 − , EpCAM − , CD31 − CAF population isolated from mouse lung and melanoma tumours inhibit T-cell function via programmed death ligands 1 and 2 (PD-L1 and PD-L2), which bind to the programmed death 1 receptor (PD-1) on T cells. 10,11 In addition to FAP + , FSP + stromal cells within ovarian carcinomas secrete factors that promote tumour growth by enhancing microvascularisation, stromal networks and protumourigenic paracrine signals. 12 Studies indicate that MSCs secrete transforming growth factor-β (TGFβ), and upregulation of the TGFβ signalling pathway in FAP + , EpCAM − , CD45 − , CD31 − CAFs within human colorectal tumours predicts metastasis and defines a poor prognosis. 13 TGFβ within the TME also promotes epithelial-to-mesenchymal transition (EMT), angiogenesis and mediates immunosuppression. [14][15][16][17] It is evident from these studies that it is difficult to distinguish between MSCs and CAFs within the TME as they express and secrete similar proteins. Nevertheless, discovering a regimen to target these tumour-associated stromal cell (TASC) populations is a key goal in cancer medicine to reduce their protumourigenic influence on growth and metastasis, and remove their block on tumour immune recognition. Indeed, ablation of FAP + TASCs reduced tumour size in mouse lung and pancreatic tumours due to increased immune control within the TME. 18,19 Additionally, ablation of tumour stromal neuron glia antigen-2 (NG2) + and PDGFRα + pericytes significantly reduces breast tumour volume in preclinical studies. 20 However, FAP + /NG2 + /PDGFRα + stromal cell depletion also causes side effects such as anaemia, cachexia and increased metastasis due to deletion of healthy stromal cells. [20][21][22] These studies confirm that TASCs play a significant role in promoting tumour growth but also highlight that safer strategies are required to inhibit TASC function so as to reduce potential side effects due to effecting normal stromal cells.
Syndecan-2 (SDC2 (human) Sdc2 (mouse)) is a heparan sulfate proteoglycan (HSPG) expressed in cells of mesenchymal origin. 23,24 Syndecan-2 structure consists of a short cytoplasmic domain, a transmembrane domain and a larger extracellular domain, which is modified towards the N-terminus with addition of heparan sulfate chains and glycosylation. 23,25 These specific functional domains enable syndecan-2 to interact with cell membrane receptors, act as coreceptors for ligand binding, as well as activate signalling pathways that promote cell adhesion and migration. [26][27][28] Syndecan-2 expression is increased in cancers of breast, pancreas, colon and prostate. [29][30][31][32][33][34] In patients with ER-negative breast cancer, high SDC2 RNA expression in breast tumours correlates with poor prognosis. 31 Additionally, inhibiting SDC2 expression in MDA-

MB-231 breast cancer cells (BCCs) reduced tumour volumes and
improved survival in an adoptive transfer mouse model of breast cancer. 31 Taken together, these studies indicate that epithelial syndecan-2 can play a pro-oncogenic role in breast cancer by promoting both tumour growth and migration. To date however, there have been no published investigations of syndecan-2 expression or function within the stromal compartment of the breast TME. In this study, we report that syndecan-2 is also expressed on the cell surface of a population of TASCs isolated from human and mouse breast tumours.
Utilising in vitro and in vivo approaches, we find stromal syndecan-2 has a key role in tumour growth, metastasis and immune evasion and is therapeutically targetable using a syndecan-2-derived peptide. were maintained in DMEM supplemented with 10% FBS, 100 U/mL penicillin and 100 mg/mL streptomycin at 37 C and 5% CO 2 . Umbilical cord, bone marrow-derived MSCs and human TASCs were maintained in α-minimum essential medium (αMEM) with 10% FBS, 100 U/mL penicillin and 100 mg/mL streptomycin with 1 ng/mL human fibroblast growth factor 2. These were cultured at 37 C, 2% O 2 and 5% CO 2 . Mouse TASCs were maintained in α-MEM with 10% FBS, 10% equine serum, 100 U/mL penicillin and 100 mg/mL streptomycin. All experiments were performed with mycoplasma-free cells.

What's new?
Tumour-associated stromal cells within the tumour microenvironment play a significant role in promoting tumorigenesis.
Current strategies to inhibit tumour-associated stromal cell function must however reduce the potential side-effects associated with affecting normal stromal cells. In this study, the authors report that Syndecan-2 is expressed on a nonepithelial, non-haematopoietic, non-endothelial stromal cell population within breast cancer tissue. Syndecan-2 contributes to the oncogenic properties of tumour-associated stromal cells by promoting TGF-β signalling, tumour growth, metastasis, and immunosuppression. Altogether, the results highlight pro-tumorigenic, immunosuppressive Syndecan-2+ stromal cells within the breast tumour microenvironment as a potential therapeutic target.

| Isolation of human TASCs
After ethical approval and written informed consent, fresh specimens of human breast tumours were harvested from patients undergoing surgery at University College Hospital Galway. Tissues were washed, minced finely and digested overnight with 0.1% collagenase type III at 37 C and 5% CO 2 . Collagenase-dissociated mammary cells were pelleted at 400g for 5 minutes and cell pellets were resuspended in 2 mL of prewarmed trypsin-EDTA by gentle pipetting and left to incubate at 37 C for 2 minutes. Trypsin was inactivated with Hanks' balanced salt solution supplemented with 2% FBS (HF). Cells were pelleted as before, resuspended in HF and filtered through a 100 μm cell strainer.
Cells were pelleted and resuspended in FACS buffer (PBS [phosphatebuffered saline] containing 2% FBS and 0.1% NaN 3 ) or stromal cell growth medium and viable cells counted using a haemocytometer. A number of 100 000 cells were incubated for 30 minutes with CD45 or syndecan-2 antibodies alone or in combination. Viability was assessed using Sytox blue staining. Data were collected using a BD FACS Canto II flow cytometer (BD Bioscience) and analysed using Flowjo software. Alternatively, cells were plated in TASC growth media and expanded as described earlier.

| Haematoxylin and eosin staining of metastatic lesions in the lung
Lungs were catheterised, perfused with 10% neutral buffered formalin and sutured shut to maintain lungs in an inflated state. They were submerged in 10% formalin for 24 hours followed by 24 hours of 100% ethanol and 24 hours of 70% ethanol. Lungs were then processed using an Excelsior AS tissue processor and embedded in paraffin wax.
Haematoxylin and eosin (H&E) staining was carried out using 6-μm tissue sections according to standard procedures. Briefly, sections were deparaffinised with xylene, rehydrated in decreasing percentages of alcohol and washed with water. Rehydrated sections were stained in Mayer's haematoxylin solution for 6 minutes. Sections were washed in running tap water for 4 minutes to undergo "blueing". Sections were counterstained with eosin for 2 minutes and rinsed in the water bath. Slides were dehydrated using a graded alcohol series ending with two changes of absolute alcohol. Slides were then cleared with xylene. Xylene was evaporated and sections were covered with Histomount xylene-based mounting solution and slides were left in a 37 C oven overnight to set.
Images were captured using a Leica bright-field inverted microscope.
Metastatic lesions were detected and each section was given a metastatic score: 1 = no visible metastatic lesion, 2 = 1 metastatic lesion, 3 = 2 metastatic lesions and 4 = 3 or more metastatic lesions.

| Generation of SDC2 fragment expression constructs and transfection
An IMAGE human cDNA clone for SDC2 (Clone ID 6383) was obtained from Open Biosystems. To generate Fc-tagged SDC2 fragment, cDNA spanning the length of amino acid 1-87 was engineered using PCR and the resulting DNA fragment was subcloned into Eco R1 and Bgl II sites within the multiple cloning site of the pFUSE-hIgG1-Fc1 expression plasmid (InvivoGen). All cloning was verified by sequencing (EuroFins).

Cell transfection was performed using FuGENE HD Nonliposomal
Transfection Reagent (Roche) using manufacturers' guidelines. In brief, 500 ng of SDC2-Fc vector or control empty vector were diluted in 100 μL of Opti-MEM Reduced Serum Media and 3 μL of FuGENE HD transfection reagent was added. The transfection mixture was mixed by gentle pipetting and left to incubate for 10 minutes at room temperature.
The transfection mixture was added dropwise to cells in a 6-well plate.

| siRNA transfection
TASCs were plated at 2 × 10 5 cells per well in a 6-well plate and allowed to adhere for 12 hours. A master mix was made per well con-

| TGFβ treatment
Cells were plated in 6-well plates at a density of 2.2 × 10 4 cells/cm 2 .
12 hours later, cells were transduced or transfected and left to incubate for 48 hours. Cells were incubated in serum-free medium for 12 hours, and TGFβ3 (R&D) was added at a concentration of 20 ng/ mL for stromal cells or 5 ng/mL for MDA-MB-231 cells. Cells were harvested at 0 and 2 hour timepoints, by first washing twice with PBS followed by addition of 350 μL RLT lysis buffer (Qiagen).

| Cumulative population doublings
TASCs were seeded into T75 flasks at a concentration of 1 × 10 5 cells per flask and allowed to grow for 7 days with media change every 2 to 3 days. After 7 days, cells were trypsinised and counted and 1 × 10 5 cells were reseeded into a T75 flask and allowed to grow for 7 days as before. This was repeated for a total of three passages and cumulative population doubling was calculated.

| RT-qPCR analysis
Total RNA was isolated using the RNeasy mini kit (Qiagen) and cDNA synthesised using a high capacity cDNA reverse transcription kit (Applied Biosystems) following manufacturers guidelines. mRNA analyses were performed using TaqMan Gene Expression Assays (Applied Biosystems). Relative quantification was performed using TATA-box binding Protein (TBP) as endogenous control.

| Colony formation assay
Cells were plated in a 6-well plate and transfected with 500 ng of SDC2 fragment -Fc DNA or empty vector control using FuGENE HD transfection reagent. 48 h later, cells were trypsinised, counted and replated at a density of 500 cells/10-cm dish. 10 days later, colonies resulting from the surviving cells were fixed, stained with crystal violet and counted.

| Migration assay
xCELLigence assays were set up following manufacturer's instructions. In brief, 160 μL of test medium was added to the bottom half of an RTCA DP CIM-Plate 16, ensuring there were no air bubbles.
The top half of the plate was fixed to the bottom and 50 μL of serum-free medium was added to the wells. Cells were prepared and washed three times in serum-free medium, cells were resuspended at 4 × 10 5 cells/mL, 100 μL of this cell suspension was added per test well, the plate was incubated at 37 C and 5% CO 2 for 30 minutes. CIM plates were inserted into the xCelligence plate and cell migration was measured every 15 minutes over a 48 hour period.    Figure 1B). 37 Additionally, cells were positive for the expression of known tumour stromal cell markers NG2, PDGFRα and podoplanin (gp38) (Supplementary Figure 1C). 38 Flow cytometry analysis demonstrates patient-derived culture-expanded TASCs express cell surface syndecan-2 to a greater extent compared to normal culture-expanded UC-MSCs ( Figure 1D). Similar to mouse breast F I G U R E 2 Legend on next page. tumours ( Figure 1A,B), syndecan-2 is expressed on the cell surface of a

| Syndecan-2 regulates TGFβ signalling, migration and immunosuppressive properties of TASCs
To date, there are no studies reporting the presence of syndecan-2 + TASCs within the breast TME or the effect of manipulating syndecan-2 within the stromal compartment of tumours, therefore we wanted to establish the role of this syndecan-2 + TASC population within the TME.
To determine this, we tested the effectiveness of two siRNA SDC2 duplexes (siSDC2-A and siSDC2-B) to reduce SDC2 levels in TASCs. Figure 2A indicates that both siRNAs effectively reduced SDC2 RNA levels. It has been previously shown that syndecan-2 is required for efficient TGFβ signalling, 28,39 therefore we examined if knockdown of SDC2 affects TGFβ signalling in TASCs. Figure 2B shows that TGFβ induces expression of Taken together, these data suggest that syndecan-2 is important for TGFβ signalling and impacts both the migratory and immunosuppressive properties of patient-derived TASCs.

| Manipulation of SDC2 in TASCs within breast tumours in vivo
To ascertain if syndecan-2 within the stromal compartment of the TME had a role in breast carcinogenesis in vivo, an orthotopic breast and PAI-1 (P < .05) ( Figure 3D). AdSDC2-transduced TASCs also enhanced lung metastasis of MDA-MB-231 cells-with increased numbers of metastatic nodules compared to those that received AdControltransduced TASCs ( Figure 3E and Supplementary Figure 3). These in vivo studies demonstrate that stromal-derived SDC2 regulates primary tumour growth and metastasis and modulates TGFβ signalling within the TME, suggesting that blocking stromal-derived syndecan-2 has the potential to inhibit tumour growth and metastasis.

| Generation of a Syndecan-2-peptide that inhibits TGFβ signalling and possesses antimigratory properties
Therefore, to develop a potential blocking agent for syndecan-2 biological activity, a SDC2 peptide fragment encompassing amino acid 1-87 was cloned into the pFUSE-human-IgG1-Fc1 vector (InvivoGen) to stabilise expression. 43 Overexpression of syndecan-2-peptide ( Figure 4A) did not affect proliferation rates (Supplementary Figure 4A) or cell viabilty (Supplementary Figure 4B) of TASCs; however, it did inhibit the migratory properties of TASCs compared to empty vector control transfected TASCs ( Figure 4B). To ascertain if SDC2-peptide affected TGFβ signalling, TASCs were treated with TGFβ and expression of TGFβ regulated genes determined. There was no difference in TGFβ-induced upregulation of SMAD7 and PAI-1 observed between SDC2-peptide-and control-transfected TASCs ( Figure 4C). However, TGFβ-induced upregulation of CXCR4 was strongly inhibited in SDC2peptide expressing TASCs ( Figure 4D). Hence similar to SDC2 knockdown, SDC2-peptide inhibits TGFβ signalling and the migratory potential of TASCs.
To determine the effect of manipulating stromal syndecan-2 with syndecan-2-peptide within the TME, the orthotopic breast cancer model described previously was used. Growth rates were compared for tumours generated with human patient-derived TASCs over- TASCs overexpressing SDC2-peptide also had lower expression of CXCR4 ( Figure 4G).

| Syndecan-2-peptide reduces the immunosuppressive properties of TASCs enabling activation of T cells
In addition to regulating TASC migration, CXCR4 contributes to the immunosuppressive properties of TASCs. 19 Additionally, PD-L1 is a TGFβ-regulated gene that controls the immunosuppressive properties of TASCs. 10,44 Therefore, we wanted to establish if similar to SDC2 knockdown, syndecan-2-peptide decreased the immunosuppressive properties of TASCs. Figure 5A shows that TASCs expressing syndecan-2-peptide expressed lower levels of PD-L1 compared to control expressing cells. Taken these data together, it would be suggested that syndecan-2-peptide causes a decrease in CXCR4 and PD-L1 expression, which has the potential to inhibit the immunosuppressive of TASCs.
To establish if syndecan-2-peptide affected the immunosuppressive properties of TASCs within the TME, we developed a syngeneic immune-competent orthotopic model whereby EO771 mouse BCCs were coimplanted with mouse TASCs into the mammary fat pad of C57BL/6 female mice at a ratio of 10:1. TASCs were transfected with syndecan-2-peptide or empty vector control. Figure 5C shows that overexpression of syndecan-2-peptide in TASCs resulted in significantly lower tumour volume by Day 9 compared to control tumours.
Tumours containing TASCs overexpressing syndecan-2-peptide also had lower levels of CXCR4 and PD-L1 expression ( Figure 5D), indicating a less immunosuppressive environment. Hence, we looked at levels of specific immune cell populations within the TME. There was no difference in the percentage of CD4 + cells, neutrophils, dendritic cells, monocytes or macrophages between syndecan-2-peptide expressing and control tumours (Supplementary Figure 5). However, there was an observed increase in the percentage of CD8 + , CD4 − , CD62L lo , CD44 mid and CD25 − -activated T cells within the TME of syndecan-2-peptide expressing tumours compared to control tumours ( Figure 5E).

| DISCUSSION
In our study, we demonstrate that syndecan-2 is expressed on the cell surface of TASCs within human and mouse breast tumours.
TASCs play a significant role in promoting tumour growth, metastasis and immunosuppression within the TME; hence, we wanted to unravel the function of syndecan-2 in TASCs. Our data indicate that syndecan-2 is important for TGFβ signalling in TASCs and regulates show TASC-derived syndecan-2 controls breast tumourigenesis via TGFβ signalling. Previous studies demonstrate that syndecan-2 inhibition (via SDC2 knockdown or syndecan-2-peptide) within the epithelial compartment of tumours also reduces tumour growth and metasatasis in immune-compromised models. 31,46 Therefore, in future studies, administration of exogenous Fc-tagged syndecan-2 peptide has the potential to be more stable in vivo, inhibit both stromal-and epithelial-derived syndecan-2 and thus be more efficacious at inhibiting tumourigenesis.
TGFβ signalling also contributes to the immunosuppressive properties of TASCs. 17 Our study demonstrates that reducing  47 In other studies, blocking the CXCL12/CXCR4 axis (AMD3100) in the stromal compartment of pancreatic and breast tumours reactivates T-cell cytotoxic capacity within the TME, thereby rendering these tumours susceptible to immune checkpoint inhibitor therapies (eg, α-PD-L1, α-PD-1 and α-CTLA-4). 19,48 This would imply due to the ability of syndecan-2 peptide to reduce stromal CXCR4 and PD-L1 expression, this gives syndecan-2 peptide the potential to render breast tumours more susceptible to cancer immunotherapies such as α-CTLA-4 antibodies, tumour-infiltrating lymphocytes (TILs) and/or chimeric-antigen receptor T cells (CAR T cells).
In summary, our data indicate that syndecan-2 is present in the stromal compartment of breast tumours and contribute to the onco-

ETHICS STATEMENT
Ethical approval was obtained from the research ethics committee at the National University of Ireland, Galway. After written informed consent, fresh specimens of human breast tumours were harvested from patients undergoing surgery at University College Hospital Galway.