Collagen induced arthritis increases secondary metastasis in MMTV-PyV MT mouse model of mammary cancer
© Roy et al; licensee BioMed Central Ltd. 2011
Received: 28 April 2011
Accepted: 22 August 2011
Published: 22 August 2011
Several studies have demonstrated that sites of chronic inflammation are often associated with the establishment and growth of various malignancies. A common inflammatory condition in humans is autoimmune arthritis (AA). Although AA and cancer are different diseases, many of the underlying processes that contribute to the disorders of the joints and connective tissue that characterize AA also affect cancer progression and metastasis. Systemically, AA can lead to cellular infiltration and inflammation of the lungs. Several studies have reported statistically significant risk ratios between AA and breast cancer. Despite this knowledge being available, there has been minimal research linking breast cancer, arthritis, and metastasis associated with breast cancer. Notably both diseases are extremely prevalent in older post-menopausal women.
To establish the novel link between arthritis induced inflammation and secondary metastasis associated with breast cancer, PyV MT mice that spontaneously develop mammary gland carcinoma were injected with Type II collagen (CII) to induce arthritis at 9 and 18 weeks of age for pre-metastatic and metastatic condition. The sites of secondary metastasis and the associated inflammatory microenvironment were evaluated.
A significant increase in breast cancer-associated secondary metastasis to the lungs and bones was observed in the arthritic versus the non-arthritic PyV MT mice along with an increase in primary tumor burden. We report significant increases in the levels of interstitial cellular infiltrates and pro-inflammatory cytokines such as interleukin-17 (IL-17), interleukin-6 (IL-6), Pro- Matrix metallopeptidase 9 (Pro-MMP9), insulin like growth factor-II (GF-II) and macrophage colony stimulating factor (M-CSF) in the arthritic lung and bone milieu as well as in the circulation. These pro-inflammatory cytokines along with the inflammatory microenvironment may be the underlying factors facilitating tumor progression and metastasis in arthritic PyV MT mice. This was further substantiated by treatment with celecoxib, an anti-inflammatory drug + αIL-17 antibody that significantly reduced the secondary metastasis to lung and bone.
The data generated not only reveal the underlying mechanism of high susceptibility to bone and lung metastasis in an arthritic condition but our combination therapies may lead to treatment modalities that will be capable of reducing tumor burden, and preventing relapse and metastasis in arthritic patients with breast cancer.
While advances have been made in breast cancer therapies, metastatic breast cancer remains an incurable disease, and thus the prevention of metastases must be a priority. The preference of breast cancer cells to grow in the bone and lung is underscored by the fact that 65-75% of patients with advanced disease develop metastasis in these organs . We hypothesize that the pro-inflammatory microenvironment within the bone and lung caused by certain inflammatory conditions may partly account for the high prevalence of secondary metastasis to those organs.
One such common inflammatory condition in humans is autoimmune arthritis (AA) which results in inflammation and deformity of the joints. Other systemic effects associated with arthritis include increased cellular infiltration and inflammation of the lungs . Although AA does not increase the risk for BC, several studies have reported that compared to cancer patients without rheumatoid arthritis (RA), those with RA have poor prognosis and higher mortality. Specifically, patients with non-Hodgkin's lymphoma, skin cancer, and BC have significantly lower survival if they suffer from RA compared to their non-arthritic counterparts [3–8].
Despite this knowledge available for a decade, it has not been fully studied in bones and lungs, the sites of chronic inflammation associated with AA, creates a milieu that attracts tumor cells to home and grow in the inflamed organs which are frequent sites of breast cancer metastasis . There has been minimal research investigating the link between breast cancer-associated metastasis and arthritis even though both diseases share several common molecular pathways of pathogenesis and both diseases are highly prevalent in post menopausal women.
We have recently shown that the incidence of breast cancer-associated bone and lung metastasis was significantly higher in mice that develop spontaneous arthritis. This was the first study that undoubtedly established a correlation between the pro-inflammatory microenvironment in bones and lungs during AA and the homing of circulating tumor cells in these sites of inflammation. Data from these studies were further substantiated in a clinically relevant model of spontaneous metastatic mammary carcinoma induced to develop arthritis. Hence, this study is a sequel of our previous study and our data corroborates a novel link between arthritis induced inflammation and secondary metastasis associated with breast cancer.
The model of spontaneous metastatic mammary gland tumors known as the MMTV-PyV MT mice carry the polyoma virus middle T-antigen driven by the mouse mammary-tumor virus promoter [10–12]. This oncogene is active throughout all stages of mammary gland development, resulting in widespread transformation and production of multifocal mammary adenocarcinomas with ~30-40% of the mice exhibiting lung metastasis by 18-26 weeks of age [10–13]. The PyV MT mice were induced to develop arthritis by administration of Type II Collagen (CII) at two time points: when the mice were 9 or 18 weeks of age designated pre-metastatic or metastatic stage respectively. The collagen-induced arthritis (CIA) model has been the most widely accepted model for inducing AA in mice. CIA is elicited in mice by immunization with CII emulsified in complete Freund's adjuvant (CFA). The ensuing pathogenesis shares several pathological features with rheumatoid arthritis (RA), including synovial hyperplasia, mononuclear cell infiltration, and cartilage degradation and the mechanism by which arthritis is induced by collagen injection in these mice is already established [14, 15].
Data clearly demonstrates a significant increase in bone and lung metastasis and decreased survival in the arthritic versus the non-arthritic PyV MT mice. In addition, we have identified some of the key proinflammatory factors in the arthritic lung and bone microenvironment and also in circulation that may contribute to the increased incidence of secondary metastasis. Further, we determined that blocking the COX-2/PGE2 and IL-17 pathways significantly reduced the formation of secondary metastasis in the PyV MT mice.
This study is of high importance with vital clinical implications, especially in the prevention of metastasis, in designing combination drug regimens, and as a diagnostic risk-assessment tool in patients with arthritis and breast cancer.
PyV MT oncogenic mice were originally a gift from Dr. W. J. Muller (McGill University, Toronto, Canada) . The PyV MT mice that we have used are congenic on the C57Bl/6 background and have been used in several of our prior publications [13, 16–19]. PCR was used to routinely identify the PyV MT oncogene. PCR was carried out as described previously . Amplification of PyV MT gene results in a 480-bp fragment.
All mice were bred and maintained in specific pathogen-free conditions in the Mayo Clinic Scottsdale Natalie Schafer Transgenic Animal Facility and UNCC Animal Facility. All experimental procedures were conducted according to Institutional Animal Care and Use Committee guidelines. All protocols were approved by the Mayo Clinic and UNCC Internal Animal Care Review Committee.
Induction of Arthritis
The PyV MT mice with spontaneous breast cancer were injected with 50 μls of 2 mg/ml CII (MD Biosciences, St. Paul, MN, USA) in CFA (Difco laboratories, Michigan, USA) intradermally ~1.5 cms distal from base of tail at two time points, at 9 weeks of age when the primary tumors are undetectable and at 18 weeks of age when the primary tumors are large enough and metastasis is expected to occur [10, 11]. Fifty-sixty percent of mice develop arthritis within 15-30 days post collagen injection and the mechanism is established.
Generation of PyV MT cells
To generate the PyV MT cell line, the tumors were dissociated in collagenase IV (1 mg/ml)(Worthington Biochemical Corporation, 730 Vassar Ave, NJ 08701) at 37°C for 30 mins. Then the tumors were meshed, cells counted and ~3 × 106 cells were plated in C-DMEM. Post 24 hours cells were replenished with fresh media.
Measurement of PGE2levels in the serum
PGE2 levels in the serum were determined as previously reported using a specific ELISA kit for PGE2 metabolite (PGEM) (13, 14-dihydro-15-keto-PGA2) (Cayman Chemicals, Ann Arbor, MI, USA) . Manufacturer's recommended protocols were followed. Serum was diluted appropriately to ensure that readings were within the limits of accurate detection. Results are expressed as picograms of PGEM/per ml. of serum.
Measurement of cytokines
The RayBio® Custom Mouse Cytokines Antibody Array kit was purchased from Ray Biotech (Norcross, GA, USA) and used according to the manufacturer's instructions. To measure the cytokines in the lung and bone microenvironment, 300 and 100 ug of protein was used respectively. Chemiluminescence was detected using an EpiChemi3® Darkroom imaging system and Lab Works® densitometry software (both from UVP Bioimaging, Upland, CA, USA). Data was corrected for background signal and normalized to positive controls using RayBio® Analysis Tool software as published (UVP Bioimaging, Upland, CA, USA).
PyV MT cells were serum-starved for 24 hrs prior to plating for the invasion assay. Cells in serum free media (50,000 cells) were plated over transwell inserts (BD Biosciences, San Jose, CA), pre-coated with reduced growth factor matrigel, and were permitted to invade towards lung lysate (300 ug protein) and bone lysate (100 ug protein) contained in the bottom chamber for 24 hours. Percent invasion was calculated as absorbance of samples/absorbance of controls × 100 .
Lungs and tumor sections were formalin fixed in 10% neutral-buffered formalin (pH 6.8-7.2) for a minimum of 24 hours. Paraffin embedded blocks was prepared by the Histology Core at The Mayo Clinic and 4-micron thick sections were cut for hematoxylin eosin (H&E) staining and for immuno-staining. Bones were decalcified using Cal-Rite (Richard Allan Scientific, Kalamazoo, MI), a formic acid decalcification agent prior to IHC. For VEGF, Pancytokeratin, and PCNA staining, sections were subjected to antigen retrieval using the DAKO Target Retrieval agent (Dako North America, Carpentaria, CA, USA). Primary antibodies to VEGF, PCNA, and pancytokeratin were purchased from Santa Cruz Biotechnologies, Santa Cruz, CA, USA and BD Biosciences, CA, USA). To determine macrophage infiltration, F4/80 antibody was purchased from Abcam, Cambridge, MA, USA. Corresponding secondary antibodies were purchased from DAKO. For all slides, 3,3" -Diaminobenzidine (Vector laboratories, Burlington, CA, USA) was used as the chromogen and hematoxylin was used as counterstain. For neutrophil staining, a standard Naphthol AS-D Chloroacetate Esterase staining using a kit from Sigma was used. (Sigma, St Louis, MO, USA; Cat: # 91C-1Kit) was used. Masson trichome staining on bone was used to determine levels of osteoclasts. All slides were examined under light microscopy and pictures taken at magnifications indicated in the Figure legend.
Western Blots and Antibodies
Equal quantities of tumor lysates were loaded on SDS-PAGE gels. COX-2 and β-actin antibodies were purchased from Santa Cruz Biotechnology, Santa Cruz, CA and were used according to manufacturer's recommendations.
Study design for the IL-17 + celecoxib treatment
To test the efficacy of anti-IL-17 antibody treatment on breast cancer-associated metastasis, PyV MT mice were injected with 50 μls of 2 mg/ml CII in CFA at 12 wks. Post 3-weeks of collagen injection, four i.p injections of 5 μg/ml of anti-IL17 antibody (BD Pharmingen, San Diego, CA) once every two weeks was administered. Celecoxib (20 mg/kg in 100 μl 10% DMSO) was gavaged  starting at the same time as the anti-IL-17 antibody but was given daily until sacrifice except for the weekends. One week after the last injection, mice were sacrificed. Controls included PyV MT mice induced with arthritis and injected with 5 μg/ml rat immunoglobulin (Ig) G1 control antibody (BD Pharmingen, San Diego, CA) in 100 μl PBS.
Image Acquisition and Analysis
Bright field images were captured from immunostains of the mouse tissue using an Olympus microscope Olympus BX60, U-ND25-2 (Olympus; Melville, NY) with magnifications (referred in Figure Legend) using the DP70 controller, processor and analysis software.
Red-green-blue (RGB)-filtered grayscale values from images from microscopic slides of mouse tissue, stained with DAB, hematoxylin or both were analyzed using the Image-Pro Plus and NIH Image processing and analyzing program . A simple translation algorithm using the RGB information was developed, providing the option for separation of DAB only- and double-stained areas from hematoxylin only-stained areas by means of subject specific thresholding, based on the correlations between the R-G- and B-filtered grayscale values. A good separation of DAB- and double-stained pixels from hematoxylin-stained pixels was achieved. Significant differences in relative areas stained and mean specific intensity for the stains between control and treatment groups in mouse tissue were tabulated. N = 3 mice and 5 fields are provided.
The densitometric analyses of immunoblots were performed using NIH Image (obtained from the NIH Web site: http://rsb.info.nih.gov/nih-image). Results are presented as mean values of arbitrary densitometric units corrected for background intensity and normalized to the expression of β-actin, or as fold increase over levels in unstimulated cells.
The Pix array 100 x-ray machine was used for bone imaging. The Pix array 100 (Bi Optic Inc, Santa Clara, CA, USA) is a commercially available x-ray machine that is designed for animal x-ray. The analysis was conducted in Carolinas Medical Center within the Department of Orthopedic Surgery.
Data were analyzed using GraphPad software (GraphPad Prism version 4.00 for Windows; GraphPad Software, San Diego, CA, http://www.graphpad.com). Results expressed as mean ± SEM and are representative of greater than or equal to three separate experiments. Comparison of groups were performed using one-way or two-way ANOVA followed by the Bonferroni post-test for multiple comparisons (*p < 0.05, **p < 0.01, ***p < 0.001). Student's t-test was used for comparing the level of significance between the experimental groups.
Decreased survival in arthritic versus non-arthritic PyV MT mice
Remodeling of the primary mammary gland tumor in arthritic PyV MT mice
Quantification of inflammation in the tumors of arthritic versus non-arthritic PyV MT mice.
Quantification in tumor
50.3 ± 19
PyV MT + CII at 9 wks
322 ± 17
PyV MT + CII at 18 wks
337 ± 22
F4/80 positive cells in the tumors of arthritic versus non-arthritic mice (5 fields from n = 3 mice were counted).
PyV MT 1
PyV MT 2
PyV MT 3
PyV MT + CII at 9 wks 1
PyV MT + CII at 9 wks 2
PyV MT + CII at 9 wks 3
PyV MT + CII at 18 wks 1
PyV MT + CII at 18 wks 2
PyV MT + CII at 18 wks 3
PCNA positive cells in the tumors of arthritic versus non-arthritic mice (5 fields from n = 3 mice were counted).
PCNA positive cells at 400× magnification
PyV MT 1
PyV MT 2
PyV MT 3
PyV MT + CII at 9 wks 1
PyV MT + CII at 9 wks 2
PyV MT + CII at 9 wks 3
PyV MT + CII at 18 wks 1
PyV MT + CII at 18 wks 2
PyV MT + CII at 18 wks 3
Quantification of VEGF staining in tumors (Average IntDen from 5 fields per tumor from n = 3 mice is shown).
Quantification in tumor
424.93 ± 19
PyV MT + CII at 9 wks
2021.8 ± 21
PyV MT + CII at 18 wks
2788.8 ± 28
Densitometry analysis of the COX-2 expression in the tumor using the image J software (data from n = 3 mice is shown)
Densitometry analysis: COX2
PyV MT 1
PyV MT 2
PyV MT 3
PyV MT + CII at 9 wks 1
PyV MT + CII at 9 wks 2
PyV MT + CII at 9 wks 3
PyV MT + CII at 18 wks 1
PyV MT + CII at 18 wks 2
PyV MT + CII at 18 wks 3
Significant increase in osteolytic metastatic lesions in the arthritic PyV MT versus non-arthritic PyV MT mice
To further confirm epithelial cell lesions, the bone sections were stained with pancytokeratin and results are shown in Figure 5G-I. Clear lesions are detected in the arthritic PyV MT bones but not in the non-arthritic PyV MT bones.
Significant increase in lung metastasis in the arthritic PyV MT mice
Significant inflammation detected in the bones and lungs of arthritic PyV MT mice
Quantification of inflammation in the bones (Average IntDen from 5 fields per bone from n = 3 mice is shown).
Quantification of inflammation in bone
C57BL/6 + CII at 9 wks
50 ± 38
C57BL/6 + CII at 18 wks
72 ± 12
PyV MT +CII at 9 wks
232 ± 15
PyV MT +CII at 18 wks
265 ± 20
To further demonstrate the chemotactic microenvironment in the lungs of arthritic versus non-arthritic mice, lung histology was examined. Moderate inflammation was noted in the C57BL/6 mice with arthritis compared to no inflammation in the non-arthritic C57BL/6 lungs. Significantly enhanced inflammation with increased cellular infiltration was observed in the lungs of PyV MT mice injected with collagen compared to PyV MT mice without collagen and compared to control C57BL/6 mice with collagen (Figure 8M-R). The pro-inflammatory phenotype in the lung correlated with the severity and incidence of lung metastasis (Figure 7) suggesting the critical role of inflammatory cells in promoting metastasis.
Enhanced invasion of PyV MT tumor cells towards arthritic bone and lung lysate
IL-17, IL-6, Pro-MMP9, IGF-II, and M-CSF may be the underlying factors responsible for the increased metastasis in the lungs and bones of arthritic mice
To determine which factors in the bone and lung microenvironment may be responsible for higher invasion, thereby driving the breast cancer cells to become more metastatic in the arthritic model, we used the RayBio® Custom Mouse Cytokines Antibody Array. The arthritic lungs and bones expressed significantly higher levels of cytokines and growth factors which included IL-17, IL-6, Pro-MMP9, IGF-II, and M-CSF (Figure 10C and 10D). This was regardless of whether the arthritis was induced at 9 or 18 wks of age suggesting that the arthritic milieu remains stable even at 10-12 weeks post CII injection. The levels of the pro-inflammatory cytokines were found to be higher in arthritic C57BL/6 lungs and bones compared to the non-arthritic C57BL/6.
Thus, we hypothesize that the pro-inflammatory microenvironment in the arthritic bone and lungs may boost the recruitment of the PyV MT tumor and that the PyV MT tumor in turn significantly augments the levels of the cytokines in these target organs thus creating a highly conducive microenvironment for the PyV MT tumors to further proliferate.
High levels of circulating PGE2coupled with increased levels of pro-inflammatory cytokines in circulation may initiate primary tumors to be more metastatic in arthritic milieu
Lastly, but expectedly, we detected significant increase in PGE2 levels in the circulation (Figure 11B). Elevated PGE2 is a hall mark of arthritis and is known to enhance primary tumor cells to become highly angiogenic and metastatic.
Treatment with anti-IL-17 and a COX-2 inhibitor significantly reduced the secondary metastasis in the arthritic PyV MT mice
Inflammation is a critical component of tumor progression and metastasis as well as arthritis [28–31]. Many processes that occur during arthritis also occur during tumorigenesis including uncontrollable growth, inflammation, increased vascularity and common cytokines and growth factors that are regulated in both. The tumor microenvironment, which is largely orchestrated by inflammatory cells, is critical in the neoplastic process, fostering proliferation, survival and migration . It is indeed interesting that post-menopausal women who are usually prone to developing some form of autoimmune arthritis including osteoarthritis, RA, or inflammatory polyarthritis are also the most likely candidates to develop breast cancer. Thus, it is not unlikely that the two diseases co-exist in these women. Although there are several studies implicating inflammation as the initiator of tumor formation and/or mediator of progression, there is minimal research on whether prior inflammation at the site of metastasis produces a fertile ground for primary tumor cells to home and proliferate. Our study begins to evaluate whether arthritis which causes inflammation in the bones and lungs enhances secondary metastasis to those sites. A significant increase in breast cancer-associated metastasis to the lungs and bones was observed in the arthritic versus the non-arthritic PyV MT mice (Figures 4, 5, 6, 7) along with increase in primary tumor burden (Figure 1). Compared with the non-arthritic C57BL/6, the lungs and bones of the arthritic C57BL/6 expresses moderate levels of inflammation even before any tumor challenge suggesting a pro-inflammatory milieu that may be responsible for attracting the PyV MT metastatic cells to the lungs and bones as demonstrated in the in vitro invasion assay (Figure 10A and 10B). Once the PyV MT cells home to the lungs or bones, the levels of interstitial cellular infiltrates and pro-inflammatory cytokines are exponentially increased which is characterized by prominent cytokines IL-17, IL-6, Pro-MMP9, IGF-II and M-CSF (Figure 10C and 10D). The levels of these cytokines along with PGE2 are also upregulated in the circulation (Figure 11A and 11B). These pro-inflammatory cytokines along with inflammatory microenvironment can facilitate tumor cell extravasation and promote metastasis. This is further substantiated when treatment with celecoxib + αIL-17 antibody significantly reduced the metastasis to lung and bone (Figure 12). This study undoubtedly authenticates our previous study .
The tumor microenvironment itself is regarded as a "smoldering" inflammation site in which many cytokines, chemokines, and enzymes mediate the inflammatory process and drive malignant progression . We observed increased inflammation into the tumor site of the arthritic PyV MT mice possibly remodeling the tumor microenvironment (Figure 2). For tumors to develop in size and amplify metastatic potential, they must make an "angiogenic switch" through perturbing the local balance of pro-angiogenic and anti-angiogenic factors. Frequently, tumors over express pro-angiogenic factors, such as PGE2 and VEGF, allowing them to make this angiogenic switch . We observed increased VEGF and COX-2 expression in the tumors of arthritic PyV MT mice compared to non-arthritic tumors (Figure 3) possibly inducing an "angiogenic switch" and contributing to invasiveness of the cells.
Cytokines and prostaglandins play an essential role in the development of arthritis . Several cytokines have been implicated in the mechanism of synovial cell activation and joint destruction in AA . At the same time, cytokines also play an imperative role in cancer development and progression. In fact, elevated serum M-CSF predicts reduced survival in metastatic breast cancer patients . At the same time, the M-CSF produced by breast cancer cells and surrounding stroma increases osteoclast formation and maturation and enhances the expression of stromal RANK ligand, both of which increase osteolytic bone degradation . M-CSF also contributes to the pathogenesis of RA through up regulation of neutrophil gelatinase-associated lipocalin (NGAL) in neutrophils, followed by induction of transitional endoplasmic reticulum ATPase (TERA), cathepsin D and transglutaminase 2(TG2) in synoviocytes . Pro-MMP9 concentration in sera and joint fluids of RA patients is reported to be significantly higher which correlates with our mouse model where the Pro-MMP9 levels are up regulated in the arthritic bone, lungs microenvironment as well as in the sera . It is reported that cathepsin G is up regulated through tumor stromal interactions and activates Pro-MMP9, active MMP9 cleaves and releases active TGF-beta, and active TGF-beta can then promote tumor growth and enhance osteoclast activation and subsequent bone resorption . Over expression of IGF-II is reported in multiple types of cancer and is proposed as a potential mechanism for cancer cells to develop resistance to IGF-1R-targeting therapy . IL-17 acts on osteoblasts by stimulating COX-2-dependent PGE2 and osteoclast differentiation factor which differentiates osteoclast progenitors into mature osteoclasts, causing bone resorption. PGE2 interacts with its eicosanoid receptors to induce the damage . It is found that synovial fluids of patients with RA contain high levels of the cytokines IL-17 and IL-15 . Cytokines play a crucial role in the regulation of inflammatory events. Inflammatory disorders such as RA are characterized by an overproduction of several cytokines including IL-6 . IL-6 on the other hand is an autocrine and paracrine growth factor for several cancers, including breast cancer [45, 46] and both IL-17 and IL-6 stimulates cancer cell growth and contributes to recurrence and metastasis in breast cancer [47–49].
The data clearly shows that breast cancer associated metastasis is increased in arthritic conditions and blocking the IL-17 and COX-2 pathways significantly reduces the development of secondary metastasis in a spontaneous model of breast cancer induced to develop arthritis.
Pinku Mukherjee, PhD, Irwin Belk Distinguished Professor of Cancer Research, Department of Biology, University of North Carolina, Charlotte, NC. Dr Mukherjee has worked on Breast Cancer for the past 22 years.
Lopamudra Das Roy, PhD, Research assistant professor, Department of Biology, University of North Carolina, Charlotte, NC. Dr Roy has received funding for her work in Breast Cancer Research from The US Department of Defense.
Sriparna Ghosh, PhD, Director, Microscopy and Imaging Core Facility, Carolinas Medical Center, Charlotte, NC. Dr Ghosh has over 10 years of experience in breast, pancreatic, multiple myeloma and testicular cancer research.
Latha Pathangey, MSc, senior technologist, Mayo Clinic Arizona, Department of Biochemistry/Molecular Biology, Scottsdale, AZ. Ms Pathangey has worked with Dr Mukherjee for five years at Mayo Clinic.
Teresa Tinder, BSc, senior technologist, Mayo Clinic Arizona, Department of Immunology, Scottsdale, AZ has worked with Dr Mukherjee for 10 years.
Helen E. Gruber, PhD, Director, Biology Division, Department of Orthopedic Surgery, Carolinas Medical Center, Charlotte, NC. Dr Gruber has over 27 years of experience in the area of bone pathology and osteoarthritis and bone metastasis.
List of abbreviations
Type II collagen
Complete Freund's adjuvant
Pro- Matrix metallopeptidase 9
Insulin like growth factor-II
Macrophage colony stimulating factor
Proliferating cell nuclear antigen
Vascular endothelial growth factor.
We are grateful to Dr Ronald J. Marler, Dr Gendler, and Dr Lee at Mayo Clinic for their valuable advice. We are grateful to all personnel at the Mayo Clinic Animal Facility for their help. We thank Natalia Zinchenko from the Carolinas Medical Center for help with bone imaging. We thank Dr Jennifer Curry for reviewing the paper. We thank Mahnaz Sahraei for immense support.
This work was funded by The Department of Defense Breast Cancer Research Program awards: BCO63396 and BC087792
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