Adipose progenitor cell secretion of GM-CSF and MMP 9 promotes a stromal and immunological microenvironment that supports breast cancer progression

A cell population with progenitor-like phenotype (CD45-CD34+) resident in human white adipose tissue (WAT) is known to promote the progression of local and metastatic breast cancer and angiogenesis. However, the molecular mechanisms of the interaction have not been elucidated. In this study, we identified two proteins that were significantly upregulated in WAT-derived progenitors after coculture with breast cancer: granulocyte macrophage colony-stimulating factor (GM-CSF) and matrix metallopeptidase 9 (MMP9). These proteins were released by WAT progenitors in xenograft and transgenic breast cancer models. GM-CSF was identified as an upstream modulator. Breast cancer-derived GM-CSF induced GM-CSF and MMP9 release from WAT progenitors, and GM-CSF knockdown in breast cancer cells neutralized the protumorigenic activity of WAT progenitors in preclinical models. GM-CSF neutralization in diet-induced obese mice significantly reduced immunosuppression, intratumor vascularization, and local and metastatic breast cancer progression. Similarly, MMP9 inhibition reduced neoplastic angiogenesis and significantly decreased local and metastatic tumor growth. Combined GM-CSF neutralization and MMP9 inhibition synergistically reduced angiogenesis and tumor progression. High-dose metformin inhibited GM-CSF and MMP9 release from WAT progenitors in in vitro and xenograft models. In obese syngeneic mice, metformin treatment mimicked the effects observed with GM-CSF neutralization and MMP9 inhibition, suggesting these proteins as new targets for metformin. These findings support the hypothesis that GM-CSF and MMP9 promote the protumorigenic effect of WAT progenitors on local and metastatic breast cancer. Cancer Res; 77(18); 5169-82. ©2017 AACR.


Introduction
Postmenopausal breast cancer is one of the leading causes of death in western countries (1).Epidemiological data indicate that in overweight/obese women breast cancer incidence is increased (2), prognosis is worsened (3), and the cancers themselves are more resistant to chemotherapy (4).White adipose tissue (WAT) promotes the growth of breast cancer in animal models, is abundant in breast tissue, and many of its cells secrete factors having paracrine and endocrine activity (5,6).In obese women adipocytes, inflammatory cells and the factors they secrete, are altered (7,8) and promote a breast microenvironment that favors cancer development, growth, migration and angiogenesis (9,10).Use of autologous adipose in breast reconstruction after mastectomy has been reported to increase the breast cancer relapse rate (11,12).It is therefore important to identify the molecules that mediate the tumor-promoting activity of WAT, not least because they may be targets for new breast cancer therapies.
WAT includes adult stem cells with progenitor-like phenotype (13,14).Progenitor cells have been found to support breast cancer growth and metastasis in preclinical models (15).Progenitors isolated from the WAT stromal vascular fraction consist of two subpopulations: mesenchymal progenitor cells or adipose stem cells (ASCs), and endothelial cells with a progenitor-like ultrastructure (ECs) (16).These have been found to have complementary effects in promoting breast cancer: ASCs support epithelial to mesenchymal transition (EMT), while ECs support local tumor growth and promote angiogenesis and metastasis (15-17).
However, notwithstanding the abundant evidence that adipocyte progenitors promote breast cancer, the molecular mechanisms mediating this effect have not been identified.In the present study we show that two proteins play key mediator roles: granulocytemacrophage colony-stimulating factor (GM-CSF) and matrix metallopeptidase 9 (MMP9).
We found that both these proteins are up-regulated in WAT progenitors exposed to breast Research.
on August 30, 2017.© 2017 American Association for Cancer cancerres.aacrjournals.orgDownloaded from Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
Author Manuscript Published OnlineFirst on July 28, 2017; DOI: 10.1158/0008-5472.CAN-17-0914 cancer cells (in vitro and preclinical models).We also found that GM-CSF produced by breast cancer cells is an up-stream inducer of the aberrant up-regulation of GM-CSF and MMP9 in WAT progenitors.
We also investigated these proteins in diet-induced obese syngeneic mouse models of breast cancer, assessing their impact on the tumor microenvironment and tumor progression.Finally we investigated the effect of metformin on this newly-identified interaction.Metformin is a widely used anti-diabetic drug recently proposed as a treatment for breast cancer, since its administration is associated with reduced cancer incidence, particularly in obese patients or with metabolic syndrome (18)(19)(20).We have shown previously that metformin efficiently reduces tumor growth, metastasis, and angiogenesis in preclinical models of breast cancer and that it targets both breast cancer cells and WAT progenitors (21). Research.
on August 30, 2017.© 2017 American Association for Cancer cancerres.aacrjournals.orgDownloaded from Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Collection and processing of WAT cells
Lipotransfer material was collected from 40-65 year old women undergoing breast reconstruction at the European Institute of Oncology, Milan, after giving informed consent.
All patient studies were conducted in accordance with the Declaration of Helsinki, and performed after approval by the Institutional Review Board.WAT cells were obtained from lipotransfer material as described elsewhere (15,16).Cells were labeled magnetically with anti-human CD45 microbeads (Miltenyi Biotec, Germany) and separated on a magnetic column (LS columns, Miltenyi Biotec).The CD45-fraction was collected and its purity assessed by flow cytometry.This fraction was then labeled with anti-human CD31 microbeads (Miltenyi Biotec) to separate ASCs (CD34+CD31-) from ECs (CD34+CD31+) (16).
All mouse studies were approved by the Italian Ministry of Health and performed in accordance with the Institutional Animal Care and Use Committee.Subcutaneous and intra-peritoneal WAT was obtained from 8-12 week-old female mice, FVB/N-Tg(MMTV-PyVT)634Mul/J (MMTV-PyMT, Jackson Laboratories, Bar Harbor, ME, US), housed in our animal facilities at the IEO-FIRC Institute, Milan.WAT was purified with anti-mouse CD45 microbeads (Miltenyi Biotec).

Cell cultures
MDA-MB-436, MDA-MB-231, HCC1937, and 4T1 breast cancer cell lines were purchased in 2014-17 from ATCC, (Manassas, VA, US), tested every six months for Mycoplasma by means of the ATCC Universal Mycoplasma Detection Kit 30-1012, expanded and stored according to the producer's instructions.Cells  than two weeks and used for no longer than 15 passages.MMTV-ErbB2+ breast cancer cells from FVB-NK1Mul/J mice were isolated and cultured as described elsewhere (22).
Quantitative reverse transcription PCR (qRT-PCR) and ELISA (#DGM00, R&D) were used to confirm knockdown in breast cancer cells, after puromycin selection.

In vivo experiments
In vivo experiments were carried out in accordance with Italian legislation and institutional guidelines.Mice were bred and housed in pathogen-free conditions.Xenografts were generated as described elsewhere (15).Six-to-eight week-old NODSCIDIL2Rγnull (NSG) female mice (12 per study arm) were injected (mammary fat pad) with 1x10 6 human breast cancer (hBC) cells (MDA-MB-436, MDA-MB-231 or HCC1937) alone or in combination with 0.2x10 6 human CD45-CD34+ WAT cells.Five mice were injected with human CD45-CD34+ cells alone as negative controls.In another set of experiments half the mice were given high-dose metformin (drinking water, 2mg/ml) three days after injection (12 per study arm) until sacrifice.Tumor growth was assessed weekly using digital calipers to determine width (W mm) and length (L mm): volume (mm 3 ) was calculated as LxW 2 /2.Serum or plasma was prepared from blood collected every two weeks from the animals' tail vein; EDTA was added to plasma.Plasma levels of hGM-CSF were determined with high sensitivity (HS) ELISA (#HSGM0, R&D).Serum levels of cholesterol, triglycerides and glucose were determined with Architect c8000 (Abbott, IL, US).When tumors reached 1.2 cm diameter, the animals were killed by CO 2 inhalation.
Five mice were administered high-dose metformin (2mg/ml in drinking water) three days after tumor injection, until sacrifice.
Tumor growth was assessed weekly.Blood was collected monthly from the tail vein, and circulating immune cells were determined by multiparametric flow cytometry (Table S1).
When tumors reached 1.2 cm the mice were either sacrificed or the tumor was removed (mastectomy).After mastectomy, the animals were sacrificed 30 days (MMTV-ErbB2+ breast cancer) or 15 days (4T1) later.Tumors, subcutaneous WAT, lungs and spleens were collected from sacrificed animals.Tissue samples were fixed in 4% phosphatebuffered formalin and embedded in paraffin.Other samples were stored for RNA (in RNAlater, Qiagen) or protein analysis (frozen in liquid nitrogen).Five-µm thick sections of lungs were stained with hematoxylin and eosin (H&E) to assess the presence of metastases.Images were acquired with a ScanScope XT scanner (Leica, Germany) and analyzed with Aperio Digital Pathology software

Genotyping
The genomic DNA of FVB/N-Tg(MMTV-PyVT)634Mul/J animals was analyzed by PCR of tail biopsies, using the Gentra Puregene Mouse Tail kit (#158267, Qiagen).Primers, internal positive controls, and cycling conditions were as recommended by Jackson Laboratory.
For Western blot analyses, frozen tissue samples were first lysed in RIPA Buffer; culture media were used directly.Total protein concentrations in samples were measured by BCA assay (Thermo Fisher Scientific).Protein concentrations were equalized by appropriate dilution and the samples (10-15µg protein) run on 7.5% Mini Protean gels (BioRad), followed by transfer to nitrocellulose membranes (Amersham, GE Healthcare) using the Trans-Blot Turbo Transfer System (BioRad).

Zymography
Culture media or whole tumor lysates (15-20µg) were loaded 1:2 with Zymogram sample Buffer (BioRad) and run on 10% Zymogram gels (BioRad).Gels were then incubated 0.5 hour in Renaturation Buffer (BioRad), followed by overnight incubation in Developmental Buffer (BioRad) at 37°C, and staining with PageBlue (Thermo Fisher Scientific) for 5 hours at room temperature.Images were acquired with ChemiDOC.qRT-PCR RNA was extracted from samples with Qiamp Mini Blood kit (Qiagen).RNA quantity and quality were checked with NanoDrop 2000 (Thermo Fisher Scientific).0.5-1µg of RNA was retro-transcribed with the Ipsogen RT kit (Qiagen).qRT-PCR was carried out on the ABI Prism 7000 platform (Thermo Fisher Scientific) using primers and probes from TaqMan Gene Expression Assays (Thermo Fisher Scientific).RT 2 Profiler PCR array for mouse inflammation and immunity cross-talk (#PAMM-181Z, Qiagen) was performed following the manufacturer's instructions.The collected data were analyzed with web-based software (RT 2 Profiler PCR Array Data Analysis; SABiosciences, Qiagen).

Flow cytometry
At least 500,000 cells per sample were acquired using a 3-laser flow cytometer (Navios, Beckman Coulter).Viable cells (negative for 7-aminoactinomycin, 7AAD) were labeled with a panel of antibodies (Beckman Coulter or BD Biosciences, San Diego, CA) to analyze immune cell populations and WAT-derived progenitors (Table S1).Lymphocytes, macrophages, granulocytes and dendritic cells were characterized using standard markers (23,24).Myeloid derived suppressor cells (MDSCs) and inflammatory monocytes were identified as a recent classification (25).Tumor-associated macrophages (TAMs) were identified according to Su et al. (26).We have previously described the complete characterization of WAT-derived progenitors, including ASCs and ECs (15,16).

Statistical analysis
Results are summarized as means and SEMs.The Shapiro-Wilk test was used to assess normality.Most data were not normally distributed so all statistical comparisons used the nonparametric Mann-Whitney U test of.All p values are two sided.Differences were considered significant for p<0.05 after Bonferroni correction.The statistical analyses were performed with GraphPad Prism software.

GM-CSF and MMP9 are up-regulated in WAT progenitors co-cultured with breast cancer cells
Co-cultures of purified WAT progenitors (CD45-CD34+) with breast cancer cell lines were analyzed for soluble factors.Two secreted molecules were found up-regulated compared to single cultures: GM-CSF and MMP9 (Fig. 1A).Up-regulation was confirmed by ELISA (GM-CSF) and Western blot (MMP9) in WAT progenitors from 15 women cultured with various human triple-negative breast cancer (TNBC) cell lines (Fig. 1B-D).TNBC cells expressed considerably higher baseline levels of GM-CSF than WAT cells cultured alone (Fig. 1B).Zymography revealed increased MMP9 activity in direct co-cultures, consistent with the greater expression found on Western blot (Fig. 1C).Transcriptional analysis showed that WAT progenitors were the primary source of GM-CSF and MMP9 in cocultures (Fig. 1E,F).Co-culture of murine WAT (mWAT) CD45-CD34+ progenitors with human breast cancer (hBC) further supported these findings since only murine transcripts were up-regulated (Fig. S1A,B).
To assess GM-CSF and MMP9 specific induction in WAT progenitors, in additional experiments we co-cultured human WAT-derived hematopoietic cells (CD45+CD34-) and WAT progenitors (CD45-CD34+) with hBC cells.The CD45+CD34-population, which constitutes the main part of the stromal vascular fraction, is increased in WAT from obese persons (9).Up-regulation of GM-CSF and MMP9 mRNA did not occur in CD45+CD34cells on exposure to breast cancer cells, but did occur in CD45-CD34+ cells (Fig. S1C).

When cultured alone ASCs and ECs did not express detectable levels of GM-CSF, but on
Although ECs expressed higher baseline levels of MMP9 than ASCs, MMP9 release was significantly increased when both progenitors were co-cultured with breast cancer cells (Fig. S1E).We also found that transcripts of GM-CSF and MMP9 were up-regulated in ECs and ASCs when co-cultured with breast cancer cells (Fig. S1F).Thus both ASCs and ECs contribute to greater production of GM-CSF and MMP9 in WAT progenitors when exposed to breast cancer cells.

Inhibition of GM-CSF and MMP9 release from co-cultured WAT progenitors
Monoclonal antibodies or inhibitors affecting putative upstream regulatory pathways, as identified in previous studies (27)(28)(29), were investigated.These were added to co-cultures to determine whether they inhibited GM-CSF or MMP9 release (Fig. 2).The NF-κB regulatory pathway was inhibited by bortezomib using concentrations that did not affect cell viability (data not shown).IL-1β signaling was blocked by anti-human IL-1β MoAb.SB-3CT (irreversible inhibitor MMP9) and anti-hGM-CSF MoAb were used to exclude reciprocal regulation.Bortezomib was found not to impair GM-CSF or MMP9 expression in co-cultures of breast cancer cells and WAT progenitors (Fig. 2A-E).However, IL-1β neutralization impaired MMP9 release in direct co-cultures (Fig. 2B) and MMP9 transcript expression in WAT progenitors (Fig. 2E).Neither IL-1β neutralization (Fig. 2A,D) nor SB-3CT (Fig. 2A) had any effect on GM-CSF release.However GM-CSF release was impaired by GM-CSF neutralization in co-cultures (Fig. 2A).GM-CSF neutralization also resulted in dose-dependent reduction in MMP9 release (Fig. 2C).qRT-PCR provided additional support for this finding since GM-CSF and MMP9 transcripts were reduced in WAT progenitors after GM-CSF neutralization (Fig. 2D,E).These results suggest that GM- CSF, produced by breast cancer cells, may be an up-stream regulator of GM-CSF/MMP9 release by WAT progenitors.

GM-CSF and MMP9 are up-regulated in xenograft and spontaneous breast cancer models
The expression of human GM-CSF (hGM-CSF) and human MMP9 (hMMP9) was assessed in xenograft models of TNBC.NSG mice were injected with TNBC cells alone or with hWAT CD45-CD34+ progenitors (Fig. S2A-C).Circulating hGM-CSF was quantified by ELISA and found to be significantly higher in co-injected (hWAT+hBC) than singleinjected (hBC) mice (Fig. 3A; Fig. S2D).Mice injected with hWAT progenitors were used as negative controls to support the specificity of the assay.hMMP9 expression was also investigated in tumors and was found at higher levels in coinjected (hBC+hWAT) than single-injected tumors (Fig. 3B; Fig. S2E).The full-length precursor (pro-MMP9, 92kDa) and cleaved (active) MMP9 forms (82kDa or 67kDa) were up-regulated in whole tumor lysates from co-injected mice, with the biologically-active 67kDa form expressed most prominently.Zymography showed that MMP9 had greater enzymatic activity in co-injected tumors (Fig. 3B).
qRT-PCR of breast cancer cells isolated from mouse tumors did not indicate significant induction of GM-CSF or MMP9 transcripts, suggesting that WAT progenitors were secreting these proteins (Fig. 3C).To provide support for this supposition, mWAT CD45-CD34+ progenitors were isolated from transgenic tumor-bearing mice (MMTV-PyMT).These cells were compared to mWAT progenitors collected from wild type (WT) mice of the same age and sex.It was found the GM-CSF and MMP9 transcripts were significantly up-regulated in WAT from transgenic mice compared to WAT from WT mice (Fig. S3A).
Protein release was also significantly greater in WAT from transgenic mice (Fig. S3B indicating that in vivo, up-regulation of these proteins in adipose progenitors depends on the presence of breast cancer.

GM-CSF knockdown in breast cancer cells prevents the pro-tumorigenic effects of WAT progenitors
GM-CSF was identified in vitro as a potential up-stream regulator of both GM-CSF and MMP9 production in WAT progenitors.To provide further evidence on this, lentiviral vectors were used to knockdown GM-CSF in the MDA-MB-436 TNBC cell line.shGM-CSF hBC cells were injected into NSG mice alone or together with hWAT progenitors.GM-CSF knockdown in tumors was demonstrated by qRT-PCR (Fig. 3D).Tumor growth in coinjected (hBC+hWAT) mice was significantly impaired when GM-CSF knockdown cells were used, compared to scramble, and did not differ from that in single-injected (hBC) mice (Fig. 3E).There were also fewer metastatic foci in lungs in GM-CSF knockdown hBC cells (Fig. 3F).These data indicate that breast cancer-derived GM-CSF supports local tumor growth and metastatic progression in preclinical models.Plasma hGM-CSF was significantly reduced after GM-CSF knockdown in both single-and co-injected mice (Fig. 3G).Thus, GM-CSF released from breast cancer cells is required to support GM-CSF production in WAT progenitors.Our data also indicate that GM-CSF is an up-stream regulator of MMP9 since we found that hMMP9 protein levels in tumor lysate were significantly lower in GM-CSF knockdown tumors compared to scramble in co-injected mice (Fig. 3H).

Combined GM-CSF neutralization and MMP9 inhibition reduce tumor growth and metastatic spread in an obese syngeneic breast cancer model
The preclinical effects of inhibiting GM-CSF and MMP9 were investigated in immunocompetent obese mice injected with TNBC (4T1 cells, in BALB/c background) or MMTV-ErbB2+ (in FVB/Hsd background).Since WAT progenitors are significantly increased (per unit weight of WAT) in obese mice (15,17), before injection of breast cancer cells, the mice were fed a HFD and rapidly gained body weight (Fig. S4A).BALB/c mice have been reported as more resistant to obesity than FVB mice, although they have similar adiposity (30).After tumor injection, the animals were administered anti-mGM-CSF MoAb, SB-3CT (MMP9 inhibitor), or both.Mouse weight was unaffected by these treatments (data not shown).Preliminary experiments (using IgG2a MoAb or vehicle as controls) showed that both anti-mGM-CSF MoAb and SB-3CT reduced tumor volume and metastatic spread in obese mice (Fig. S4B,C).Administration of GM-CSF and MMP9 inhibitors together revealed they acted synergistically to reduce local breast tumor growth in MMTV-ErbB2+ breast cancer (Fig. 4A).Metastatic spread to lungs was investigated 30 days after mastectomy.Administration of both inhibitors together had the best efficacy in reducing metastatic spread (p<0.001,Fig. 4B).
In the other obese model of breast cancer (4T1-injected Balb/c) tumor growth was significantly reduced by GM-CSF neutralization and MMP9 inhibition (p<0.001,Fig. 4C).
SB-3CT was the most effective in reducing lung metastases in this model (p<0.01,Fig. 4D).Spleens from these mice were less enlarged (lower weight) than those from IgG2a+vehicle controls (p<0.01,Fig. 4E).These findings indicate that GM-CSF and MMP9 are involved in the local and metastatic growth of triple negative and ErbB2 overexpressing breast cancers in diet-induced obese mice.

GM-CSF neutralization and MMP9 inhibition impair neoplastic angiogenesis in vivo
GM-CSF and MMP9 are known modulators of angiogenesis.GM-CSF influences angiogenesis by regulating the coordinated expression of VEGF (31); MMP9 triggers the angiogenic switch during carcinogenesis (32).To investigate whether angiogenesis is affected by GM-CSF neutralization or MMP9 inhibition, we performed double immunefluorescence analysis, staining endothelial cells (CD31+ cells) and pericytes (αSMA+ cells) in tumors from obese mice.In both models, GM-CSF neutralization and MMP9 inhibition resulted in strong inhibition of intratumoral angiogenesis, with significantly (p<0.01)lower microvessel density (MVD) (Fig. 5A-C).Use of both inhibitors together further impaired tumor angiogenesis, suggesting that both factors are crucially involved in breast cancer neovascularization.Use of both inhibitors did not affect the ratio of αSMA+ to CD31+ blood vessels, but did preferentially target αSMA+ cells (Fig. 5D).

GM-CSF promotes an immunosuppressive microenvironment, possibly leading to tumor immune escape
Multiparametric flow cytometry was used to investigate immune cells (for gating strategy see Fig. S4D).Peripheral blood, tumors, and peritumoral WAT were collected from obese tumor-bearing mice.A month after starting GM-CSF neutralization, the number of circulating monocytes was found to be significantly lowered in GM-CSF-neutralized mice, compared to controls (CTR) administered IgG2a MoAb (Fig. 5E).Immunosuppressive cells were also significantly reduced by GM-CSF neutralization in tumors and peritumoral WAT: T-regulatory (T-reg) cells and granulocytic (G)-MDSCs were reduced in tumors and WAT (Fig. 5F-G), whereas monocytic (M)-MDSCs were reduced in tumors only (Fig. 5F).
Macrophage and TAM infiltration was also significantly reduced by GM-CSF neutralization.Taken together, these findings suggest that GM-CSF is able to promote the recruitment of immunosuppressive cells to the tumor microenvironment, to thereby promote immune escape by tumor cells.By contrast, MMP9 inhibition (SB-3CT) had no effect on immune cells in peripheral blood, WAT or tumors (data not shown).
To further investigate immunosuppression, WAT and tumor cells from GM-CSFneutralized mice were analyzed by qRT-PCR.In comparison to IgG2a controls, transcripts of several immunosuppressive factors were downregulated, including IL-10, IL-5, CXCL5, CCL22, CCL4, CXCR5 and CD274 (PD-L1) (Fig. S4E).GM-CSF neutralization more profoundly reduced the expression of these genes in WAT than tumor cells.It is noteworthy that GM-CSF and IL-1β gene expression was also downregulated in WAT and tumors, providing in vivo support to the GM-CSF regulation observed in vitro.

Metformin inhibits GM-CSF and MMP9 release in vitro
The antidiabetic drug metformin has been reported to inhibit the onset of several breast cancer subtypes in diabetic patients, especially those who are obese (18,19).The drug targets neoplastic and microenvironment cells, including endothelial cells and WATderived progenitors (17,21).Metformin was added directly to in vitro co-cultures to determine whether it affects GM-CSF and MMP9 release.Metformin was added at the concentration 5mMa level at which cells remained viable at 72 hours (Fig. S5A,B).GM-CSF and MMP9 release was reduced in co-culture media to which metformin had been added, but not in single cell cultures (Fig. 6A,B).As expected, zymography showed reduced MMP9 enzymatic activity in the presence of metformin (Fig. 6C).GM-CSF and MMP9 transcript levels were unaffected by metformin (data not shown), suggesting that metformin does not affect the expression of these proteins at the transcriptional level.

Metformin reduces hGM-CSF and hMMP9 expression in xenograft models
Xenograft NSG mice, injected with hBC cells alone or with CD45-CD34+ hWAT progenitors, were given metformin in drinking water (2mg/ml) at a higher dosage than that used in diabetic patients, but found to be effective and non-toxic in preclinical models of breast cancer (21).Neither mouse weight, nor serum levels of glucose, cholesterol or triglycerides were significantly affected by the drug (data not shown).As expected in this model (21), tumor growth was significantly reduced (Fig. S5C).Plasma levels of hGM-CSF were reduced in metformin-administered mice, compared to controls (Fig. 6D).hMMP9 expression in whole tumor lysates, previously shown to be up-regulated in hBC-hWAT coinjected mice, was reduced in metformin-treated mice (Fig. 6E).By contrast, metformin did not reduce hMMP9 expression in tumor lysates from single-injected (hBC) mice, in fact pro-MMP9 levels were higher in metformin-administered mice (Fig. 6E).

Metformin reduces angiogenesis and breast cancer progression in obese syngeneic models
Since metformin reduces GM-CSF and MMP9 release in vitro and in vivo, we investigated the effect of the drug in obese syngeneic models of breast cancer.Mice were administered either metformin, anti-mGM-CSF MoAb or SB-3CT (Fig. 7).These treatments significantly reduced local breast cancer growth (tumor volume) compared to control, with GM-CSFneutralization achieving a greater (not significant) reduction (Fig. 7A).Metastases in lungs were significantly reduced in treated mice (p<0.01,Fig. 7B).
Quantification of intratumoral CD31+αSMA+ blood vessels indicated that metformin was more effective (p<0.001) in reducing angiogenesis than GM-CSF neutralization or MMP9 inhibition (p<0.01) (Fig. 7C).Flow cytometry of tumors and peritumoral WAT indicated that immunosuppressive populations, including T-regs and TAMs, were downregulated in peritumoral WAT, whereas G-MDSCs were strongly downregulated in tumors.Metformin significantly reduced the presence of inflammatory monocytes in both tumors and peritumoral WAT, providing further evidence of its anti-inflammatory activity.These findings suggest that GM-CSF and MMP9 might be previously unrecognized targets of metformin, which would explain some of its anti-tumor effects in breast cancer preclinical models.

Discussion
We investigated interactions between breast cancer cells and CD45-CD34+ WAT progenitors because the latter promote tumor growth, metastasis and angiogenesis in preclinical breast cancer models (15,16).We found that the proteins GM-CSF and MMP9 were significantly upregulated in murine and human WAT progenitors on exposure to a variety of breast cancer cell types in vitro and in vivo.These proteins were not upregulated in other WAT cells (e.g.hematopoietic cells).Both ASCs and ECsconstituents of the CD45-CD34+ WAT fractionproduced GM-CSF and MMP9 when co-cultured with breast cancer cells, consistent with previous findings that ASCs and ECs cooperate to support breast cancer growth, angiogenesis and metastatic spread (16).
GM-CSF is a growth factor for hematopoietic and immune cells, that mobilizes stem cells and induces macrophage/granulocyte differentiation (33).Other roles include the regulation of inflammation and autoimmunity (34).MMP9 is a type IV collagenase whose increased expression has been reported associated with higher grade, metastasis, and angiogenesis in several cancers (29,35).
We investigated various pathways to clarify breast cancer-dependent GM-CSF/MMP9 upregulation.Our findings indicate that GM-CSF produced by breast cancer cells induces GM-CSF and MMP9 release from WAT progenitors; IL-1β might be also induced by GM-CSF, as our and previous data suggest (28), possibly implicating it in MMP9 release by WAT progenitors.
In mice co-injected with breast cancer cells and WAT progenitors we found greater tumor volume and more metastases than in mice injected with breast cancer cells alone.
However, when GM-CSF was knocked down in breast cancer cells, there was no increase in tumor growth.Thus tumor-derived GM-CSF seems to be essential for triggering the protumorigenic actions of WAT-progenitors.Other studies support the existence of such regulation.Thus, GM-CSF has been reported to up-regulate MMP9 in squamous cell carcinoma (29), and GM-CSF feedback regulation has been reported in myeloid cells (36).
Considering that CD116, the GM-CSF receptor, is expressed on myeloid cells (36), we are now investigating why WAT progenitors do not produce GM-CSF and MMP9 in isolation but only when co-cultured, and the role of CD116 expression in obesity and WATembedded tumors.
To further explore the roles of GM-CSF and MMP9 in promoting breast cancer we examined the effects of inhibiting these proteins in a syngeneic breast cancer model generated in immune-competent obese mice.Obese mice were used because WAT progenitors are markedly increased in adipose tissue in obesity (17).Use of immunecompetent animals allowed us to investigate the effect of GM-CSF on immune cells.We found that both GM-CSF neutralization and MMP9 inhibition significantly reduced tumor volume and number of metastases.However these anti-tumor effects may not be due (entirely) to direct interaction with tumor cells, but to microenvironment modulation, since GM-CSF neutralization reduced angiogenesis and immunosuppressive cells within the tumor, and MMP9 inhibition reduced angiogenesis and metastasis.MMP9 may exert its cancer-promoting effects by multiple mechanisms including degrading basement membranes (35), thus MMP9 inhibition could reduce tumor invasion into surrounding tissues.Some recent data suggest that while angiogenesis inhibitors may suppress growth of primary tumors, they might also push the tumor into a more aggressive metastatic state (37).Accordingly, more studies in different models would be useful to better investigate the role of GM-CSF and MMP9 blockade in cancer local and metastatic progression.
Myeloid cells are known to be targeted by GM-CSF, which has been identified as a macrophage chemoattractant in the presence of WAT inflammation (28).In our models, GM-CSF neutralization was associated with reduced numbers of monocytes in PB, and reduced macrophages and MDSCs in tumors and peritumoral WAT, while TAMs were reduced in tumors (TNBC model) and also peritumural WAT − supporting the finding of Su et al. (26) that GM-CSF as a TAM inducer in breast cancer.Macrophage accumulation has been found to promote tumor growth in obesity by increasing chronic inflammation, immune escape, and angiogenesis (38).Thus it is possible that some of the anti-tumor effects of GM-CSF neutralization found in our study might be due to reduced macrophage activation.We note also that GM-CSF can expand MDSCs in breast cancer (39).
Lymphoid cells were not affected by GM-CSF neutralization in our experiments, consistent with reported lack of expression of GM-CSF receptors on T-cells, B-cells and natural killer cells (40).Nevertheless, T-regs were downregulated in GM-CSF-neutralized mice, so GM-CSF may modulate these cells, as suggested elsewhere (41).Several immunosuppressive interleukins and cytokines have also been reported downregulated in WAT and tumors from GM-CSF-neutralized mice, including IL-10, CCL22 and PD-L1 (42)(43).
The prominent pro-tumorigenic effect of GM-CSF found in our obese syngeneic models is consistent with recent findings that GM-CSF ablation in in vitro primary pancreatic cancer cells resulted in defeated immune escape (44).Nevertheless the tumor-promoting effects of GM-CSF are surprising when considered alongside current therapeutic applications of GM-CSF such as hematopoietic stem cell mobilization (45) and immunotherapy (46).However many GM-CSF effects are dose-and context-dependent (47), Eubank et al (48) have reported opposite effects of GM-CSF in a different breast cancer model, thus the quantity of GM-CSF present in the tumor microenvironment may determine its biological effects on WAT progenitors.
As regards MMP9 inhibition, achieved by administration of SB-3CT, we found that this was associated with significant reduction in tumor progression in obese preclinical models.
However, in general MMP inhibitors have failed to produce beneficial effects in cancer patients (49).This may be due to enrollment of patients with late-stage disease, whereas MMP9 inhibition might be more efficient in early stage disease or in the presence of concomitant obesity.
We used metformin to disrupt the interaction between WAT progenitors and breast cancer cells.We and others have shown that metformin delays tumor progression by effects on both breast cancer cells and WAT progenitors (17,21).Metformin also reduces hyperglycemia and hyperinsulinemia, which are frequently associated with inflammation of breast WAT and breast cancer in humans (50).We found that metformin significantly reduced GM-CSF and MMP9 release in co-cultures, but not in single cell cultures, without affecting transcription.In vivo, in xenograft mice, high-dose metformin reduced levels of hGM-CSF and hMMP9 proteins.Metformin probably reduced the translation of these proteins in association with mTOR inhibition and decreased phosphorylation of S6 kinase (51).When we administered high-dose metformin to obese breast cancer models, it significantly reduced local tumor growth and metastasis, in a similar way to GM-CSF/MMP9 inhibition.The drug was also effective in reducing intratumoral angiogenesis and intratumoral immunosuppressive cells (TAMs, G-MDSCs, T-regs).Metformin blocks GM-CSF and MMP9 production only in co-cultured cells.As there is an evidence thatdepending upon models and drug dosagemetformin can target a variety of pathways other than mTOR (17,21,(50)(51) , we are currently investigating multiple potential effects of metformin in the presence or in the absence of concomitant GM-CSF or MMP9 blockade.
Overall, these findings suggest that GM-CSF and MMP9 may be new targets of metformin in breast cancer, and add support to use of the drug in clinical studies on breast cancer, particularly in women who are obese or have insulin resistance.Nevertheless, further studies are required to clarify the mechanisms by which metformin prevents GM-CSF/MMP9 up-regulation in WAT progenitors.Although in our model GM-CSF/MMP9 blockade had an effect also in WAT-poor organs such as the lungs, it would be crucial to further investigate how metformin and GM-CSF/MMP9 blockade can be effective in metastases arising in organs where there is little or no WAT, likefor instance -the brain or the bone, and how metformin and GM-CSF/MMP9 blockade can be effective in inhibiting primary BC cells collected from these metastatic sites.Another relevant field for investigation will involve the study of the protumorigenic role of WAT cells from mice of different ages and different strains.

Figure 7
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