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. Author manuscript; available in PMC: 2012 Jan 1.
Published in final edited form as: Cancer. 2010 Aug 30;117(1):207–217. doi: 10.1002/cncr.25563

ELEVATED EXPRESSION OF CXC CHEMOKINES IN PEDIATRIC OSTEOSARCOMA PATIENTS

Yiting Li 1, Ricardo Flores 1, Alexander Yu 1, M Fatih Okcu 1,3, Jeffrey Murray 4, Murali Chintagumpala 1,3, John Hicks 2,3, Ching C Lau 1,3, Tsz-Kwong Man 1,3,#
PMCID: PMC2997117  NIHMSID: NIHMS222451  PMID: 20806347

Abstract

BACKGROUND

Osteosarcoma is the most common malignant bone tumor in children. Despite the advent of chemotherapy, the survival of osteosarcoma patients has not been significantly improved recently. Chemokines are a group of signaling molecules that have been implicated in tumorigenesis and metastasis.

METHODS

We used an antibody microarray to identify chemokines that were elevated in the plasma samples of osteosarcoma patients. The results were validated using ELISAs on an independent set of samples. The tumor expressions of three chemokines were examined in two sets of osteosarcoma tissue arrays. We also evaluated the proliferative effect of the chemokines in four osteosarcoma cell lines.

RESULTS

We found that the plasma levels of CXCL4, CXCL6 and CXCL12 in the osteosarcoma patients were significantly higher than the controls, and the results were validated by an independent osteosarcoma cohort (p < 0.05). However, only CXCL4 (100%) and CXCL6 (91%) were frequently expressed in osteosarcoma, whereas CXCL12 was only expressed in 4%. Survival analysis further showed that higher circulating levels of CXCL4 and CXCL6, but not CXCL12, were associated with a poorer outcome of osteosarcoma patients. Addition of exogenous chemokines significantly promoted the growth of different osteosarcoma cells (p < 0.05).

CONCLUSIONS

Our results demonstrate that CXCL4 and CXCL6 are frequently expressed in osteosarcoma and the plasma levels of these two chemokines are associated with patient outcomes. Further study of these circulating chemokines may provide a promising approach for prognostication of osteosarcoma. Targeting these chemokines or their receptors may also lead to a novel therapeutic invention.

Keywords: osteosarcoma, CXC chemokines, biomarkers, antibody microarray, cell proliferation

Introduction

Osteosarcoma (OS) is a primary malignant tumor of bone arising from primitive bone-forming mesenchymal cells1. OS is the most common malignant bone tumor in children and adolescents with an annual incidence rate of 5.6 per million, and accounts for 60% of pediatric bone tumors2. OS is a devastating disease characterized by a high local aggressiveness and tendency to metastasize to the lungs and distant bones. Despite recent advances in multimodality treatments consisting of adjuvant chemotherapy and surgical resection, pulmonary metastasis occurs in approximately 40–50% of the patients. In such cases, the overall 5-year survival rate is only 28%3,4. Previous studies have found specific populations that are at higher risks of developing OS, such as patients harboring certain gene mutations, such as TP535, RB6, or RECQL47. Identification of tumor-derived factors that are associated with OS will likely improve the diagnosis and treatment of this deadly disease.

Cytokines are secreted or membrane-bound proteins that are released in response to a diverse range of cellular stresses including inflammation and malignancy. These proteins regulate the growth, differentiation and activation of immune cells in response to specific cellular stimuli. Chemokines are a family of cytokine-like proteins that selectively attract and activate different cell types including immune cells. By interacting with G-protein-coupled receptors, chemokines can affect various cellular processes, such as cytoskeleton rearrangement, directional cell migration, and cell adhesion8,9. Some chemokines can promote proliferation, angiogenesis, metastasis, or immunology suppression on cancer, while other chemokines inhibit tumor-mediated angiogenesis and promote anti-tumor immune responses9-12. CXC chemokines contain two highly conserved cysteine residues separated by a nonconserved amino-acid residue (Cysteine-X-Cysteine sequence) at the N-terminus15. The CXC chemokines are small proteins with a functional ability of activating and directing chemotaxis of different cells including leukocytes16. In OS, addition of a CXCR4 inhibitor or antagonist can significantly inhibit the development of lung metastasis in mouse models13,14. In this study, we tested if other cytokines and/or chemokines were also associated with OS.

MATERIALS AND METHODS

Patients and Samples

The sample and patient information used in this study is listed in Table 1. The human plasma samples were collected at initial diagnosis from 33 OS patients who were enrolled from three collaborating institutions, namely Texas Children's Hospital (TCH, Houston, TX), Cook Children’s Medical Center (Fort Worth, TX), and the University of Oklahoma Health Sciences Center (Oklahoma City, OK). Patients were 7 to 22 years of age at diagnosis. Twenty-one plasma samples from anonymized hospitalized pediatric patients with non-cancerous diseases (well-child checkup, flu, constipation, gastroenteritis, and febrile seizure) were collected from TCH and used as non-cancerous disease controls (D-CTL). All patients gave consent to Institutional Review Board-approved protocols. All plasma samples were collected in EDTA-containing tubes at room temperature and immediately centrifuged at 1000 rpm for 10 min. The plasma supernatant was stored at 80°C until use. Five human plasma samples from healthy 18-year-old donors were used as healthy controls (N-CTL) in the ELISA validation (Equitech-Bio, Inc, Kerrville, TX).

Table 1.

Sample and patient information used in the study A. Peripheral blood samples used in the study

A. Peripheral blood samples used in the study
Sample set Group Description Number Use
Discovery OS Osteosarcoma 33 Identification of chemokines using RayBio Antibody Array and ELISAs of three CXC chemokines
D-CTL Non-cancerous controls 21
N-CTL Healthy controls 5 ELISAs of three CXC chemokines
Validation OS Osteosarcoma 51 ELISAs of three CXC chemokines
N-CTL Healthy controls 11
B. Clinical characteristics of OS patients
Characteristics All patients Training set Validation set
Total number 84 33 51
Age at diagnosis (Year)
 Median (range) 13 (4–22) 13 (7–22) 13 (4–22)
 <=10 19 (23%) 5 (15%) 14 (27%)
 11–17 58 (69%) 25 (76%) 33 (65%)
 >=18 7 (8%) 3 (9%) 4 (8%)
Gender
 Male 45 (54%) 17 (52%) 28 (55%)
 Female 39 (46%) 16 (48%) 23 (45%)
Primary site
 Extremities 79 (94%) 31 (94%) 48 (94%)
 Others 5 (6%) 2 (6%)* 3 (6%)#
Metastasis at diagnosis
 Yes 18 (21%) 8 (24%) 10 (19%)
 No 66 (79%) 25 (76%) 41 (80%)
Survival
 Died of disease 10 (12%) 10 (30%) 0 (0%)
 Alive 21 (25%) 21 (64%) 0 (0%)
 NA 53 (63%) 2 (6%) 51 (100%)
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*

The other primary sites of OS in the discovery set include pelvis and palate.

#

The other primary sites of OS in the validation set include shoulder, pelvis, and nose base.

NA denotes the information is not available.

Serum samples used in an independent test set were collected by the Children’s Oncology Group (COG) under the protocol P9581 from multiple institutions. All 51 serum samples used in this study were collected at the time of diagnosis from OS patients who were 4 to 22 years of age (Table 1). Nine serum samples from 18-years-old healthy donors (Equitech-Bio, Inc.) were also used as N-CTLs in the validation phase. The serum and plasma samples were analyzed by SDS PAGE and the quality and protein patterns between the two sets of samples were very similar (data not shown).

RayBio Antibody Array analysis

Biotin Label-based Human Antibody Array I (RayBiotech, Inc., Norcross, GA) was used to detect 507 human proteins in plasma as in the manufacturer’s manual. In this study, 33 OS and 21 D-CTL plasma samples were pooled separately and hybridized with the array. The spot signals of were detected using ScanArray® Express Microarray Scanner (PerkinElmer, Inc., Waltham, MA). Duplicated experiments were performed for each sample. The median minus background intensities of all the experiments were log-2 transformed and quantile-normalized. The intra-array coefficients of variation (CVs) ranged from 9.8% to 13.9%. The correlation coefficients of the spot intensities in the replicate experiments of the control and the tumor samples were 0.94 and 0.93, respectively (data not shown), suggesting that the data generated from the array platform were reproducible. Intensity ratios between OS and D-CTL of each experiment were used to identify candidate biomarkers. The p-values were calculated using 2-sample t-test. In the antibody study, the candidate biomarkers were identified with a p-value < 0.005 and fold change > 1.4 to adjust for multiple testing.

Enzyme-linked Immunosorbent Assay (ELISA) Verification and Validation

The CXCL4 concentrations were determined using the IMUCLONE Platelet Factor 4 ELISA kit (America Diagnostica Inc., Stamford, CT). The CXCL6 and CXCL12 concentrations were determined using Human GCP-2/CXCL6 and CXCL12/SDF-1 alpha Quantikine Immunoassay kits (R&D Systems, Inc., Minneapolis, MN), respectively. The intensity values were analyzed using 2-sample t-test. A p-value < 0.05 was considered to be significant. The survival analysis was performed using the Kaplan-Meier estimation and the significance was calculated by the log-rank test using the statistical software SPSS (Chicago, SPSS, Inc). The cutoffs for the survival analysis were based on the second or third quartile of the chemokine concentration data from the OS patients. The quartile was chosen based on manual inspection to identify the best separation of the survival groups.

Immunohistochemistry (IHC)

Two sets of paraffin embedded OS tissue arrays were used in this study. One array contains 49 OS and 28 chondrosarcoma (CS) cases (Biomax, Inc., Rockville, MD). The other OS tissue array contains 64 OS and 10 rhabdomyosarcoma (RMS) cases (COG). The staining was performed using VECTASTAIN ABC system (Vector Laboratories, Burlingame, CA). Each of the mouse anti-human monoclonal primary antibodies for CXCL4 (Abcam, Inc., Cambridge, MA), CXCL6, and CXCL12 (R&D Systems) was diluted to 10 μg/ml. Immunostaining score was calculated based on the sum of the proportion score of positive cells and the intensity score of the staining by the pathologist (JH). The proportion score of positive cells was classified as 0 when <1% of stained cells were observed within the tumor, 1 when 1–25% of the tumor cells were positive, 2 when 25–50% of the tumor cells were positive, 3 when 50–75% of the tumor cells were positive, and 4 when >75% of the tumor cells were positive. The intensity score was classified as 0, 1, 2, and 3 for trace staining (background or minimal), weak staining, moderate staining, and strong staining, respectively. The results of staining in tumor cells were subdivided into three groups as follows: negative if scored 0, weak if scored 1 to 4, and strong if scored 5 to 7.

Proliferation assay

OS cell lines MG-63, U2-OS, SaOS-2, and SJSA were purchased from American Type Culture Collection (Manassas, VA). Cells were cultured at 37°C in DMEM medium supplemented with 10% fetal bovine serum (Invitrogen Corp., Carlsbad, CA). Cell proliferation was measured by Cell Counting Kit-8 (Dojindo Molecular Technologies, Inc., Rockville, MD). For each OS cell line, six replicates of 5000 cells were seeded into 96-well flat-bottomed plates in DMEM without fetal bovine serum. Each of the recombinant human CXCL4, CXCL6, and CXCL12 (R&D Systems) was reconstituted in phosphate buffered saline containing 0.2% human serum albumin. After overnight culture, cells were treated with 1 μg/ml of CXCL4, CXCL6, or CXCL12 for 24 hours. Then 10 μl of CCK-8 in 100 μl of the medium were added to each well for 3 hours. The absorbance of each of the reaction was measured at the wavelength of 450 nm.

RESULTS

Identification of Circulating Biomarkers for OS Using Antibody Arrays

To identify OS-associated chemokines or cytokines, we analyzed a pooled OS sample and a pooled disease control (D-CTL) sample on antibody arrays (Table 1A). By measuring the signals of the 507 proteins on the array, we identified 20 differentially abundant proteins in the plasma of OS patients when compared with those in D-CTL. Among these 20 proteins, eight were higher and 12 were lower in the OS patients (Table 2). Interestingly, three of the eight elevated proteins were members of the CXC chemokine family. They were CXCL4 or platelet factor 4 (OS/D-CTL = 1.7), CXCL6 or granulocyte chemotactic protein-2 (OS/D-CTL = 2.1), and CXCL12 or stromal cell derived factor -1 (OS/D-CTL = 1.4) (Fig. 1A). Ten other CXC chemokines detected by the array (CXCL1, CXCL5, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL13, CXCL14, and CXCL16) did not show any differential abundance between the OS patients and the D-CTL.

Table 2.

Differentially abundant proteins identified in the RayBio Antibody Arrays.

No. Protein Name OS/D-CTL Ratio p-value
Increased in OS 1 CXCL6/GCP-2* 2.1 0.000006
2 TGF-beta RIII 1.8 0.00002
3 CXCL4/PF4* 1.7 0.0006
4 MIF 1.5 0.0002
5 PARC/CCL18 1.5 0.0004
6 IFN-alpha / beta R1 1.4 0.0006
7 CXCL12/SDF-1* 1.4 0.001
8 M-CSF 1.4 0.001
Decreased in OS 1 Smad 4 0.00008
2 NRG2 2.1 0.00003
3 MMP 1.8 0.00004
4 MUSK 1.7 0.0002
5 NRG3 1.6 0.000006
6 Frizzled-3 1.5 0.0009
7 MIP2 1.5 0.00003
8 IL-9 1.5 0.00006
9 FGF-19 1.5 0.0002
10 GFR alpha-2 1.5 0.0002
11 CD163 1.4 0.0002
12 Dtk 1.4 0.0001
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*

The three CXC chemokines were selected for the validation study. p-values were calculated using 2-sample t-test.

Fig. 1.

Fig. 1

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Elevation of the three CXC chemokines in the peripheral blood samples of the OS patients. A. ELISAs of the three chemokines in plasma samples from the discovery phase. B. ELISAs of the three chemokines in the serum samples from the validation phase. * and ** denote p < 0.05 and p < 0.01, respectively.

Validation of the Three CXC Chemokines

To confirm the antibody array results, we used ELISAs to measure the concentrations of the three chemokines in each of the plasma samples. We also included five plasma samples from 18-year-old healthy donors (N-CTL) to determine the baseline levels of these three chemokines in normal and healthy individuals. The ELISA results showed that higher concentrations of the three chemokines were significantly associated with OS patients relative to both D-CTL and N-CTL (p < 0.05, Fig. 1A). For instance, CXCL4 in the OS samples was 2.8-fold and 69.8-fold higher than the D-CTL and N-CTL, respectively (Table 3). We also observed that the levels of CXCL4 and CXCL6, but not CXCL12, in the D-CTL were higher than the N-CTL, suggesting that these two chemokines may also react to other non-cancerous conditions. Nonetheless, the two chemokines in the OS samples were significantly higher than the D-CTL samples (p < 0.05, Table 3).

Table 3.

Elevated levels of the three CXC chemokines in the OS peripheral blood samples used the discovery and validation experiments.

Protein Ratio/ p-value Antibody Array
 ELISA of the Discovery Set
 ELISA of the Validation Set
OS/ D-CTL OS/ D-CTL D-CTL/ N-CTL OS/ N-CTL OS/ N-CTL
CXCL4 Ratio 1.7 2.8 25.1 69.8 62.5
p-value 7 × 10−5 0.04 0.003 2 × 10−7
CXCL6 Ratio 2.1 1.8 8 14.4 13.6
p-value 0.036 0.139 0.011 4× 10−8
CXCL12 Ratio 1.4 1.4 0.9 1.2 1.5
p-value 5 × 10−6 0.16 0.03 5 × 10−6
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p-values were calculated using 2-sample t-test.

Next, we tested if we could validate the chemokine results in an independent test set of OS patients obtained from the Children’s Oncology Group (COG). A comparison between 52 OS and 11 N-CTL serum samples showed that all three chemokines were significantly higher in the OS patients relative to the controls (p < 0.05, Fig. 1B). For instance, CXCL6 was 62.5-fold higher in the OS samples when compared to the N-CTL. Although the equivalent of D-CTL samples was not available for the validation study, the fold changes of the three chemokines between OS and N-CTL in the discovery and validation sets were very consistent (Table 3).

Expression of the Three Chemokines in OS Tissues

We tested if these circulating chemokines play a direct role in the tumor by examining their expressions in OS tissues on two sets of OS tissue arrays. The first OS tissue array (Biomax) contains 49 OS and 28 CS cases (Fig. 2). The second OS tissue array (COG) contains 64 OS and 10 RMS cases (Fig.2). For CXCL4, 82% and 88% of OS cases had a strong CXCL4 expression in the Biomax and COG arrays, respectively (Table 4A). Most of the strong expression cases were due to high proportions of positive cells (Proportion score of positive cells = 3–4) with a medium to high expression of CXCL4 (Intensity score = 2–3). (Table 4B). We also observed a high expression of CXCL4 in all 10 RMS cases tested; however, only 25% of the CS cases highly expressed CXCL4. For CXCL6, 80% and 100% of OS cases showed a positive staining (score 1–7) in the Biomax and COG arrays, respectively. All the RMS cases but only 32% of CS cases showed a positive staining of CXCL6. It is also interesting to note that a large proportion (82%) of OS cases in the COG array exhibited a high expression of CXCL6 (score 5–7). In contrast to the other two chemokines, CXCL12 was not expressed in all RMS and CS cases, and only a few OS cases showed a positive staining (Table 4A).

Fig. 2.

Fig. 2

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Representative immunohistochemistry (IHC) results of the three CXC chemokines on the two sets of OS tissue microarrays (400X). A. IHC of CXCL4: A1, OS (Score = 7); A2, CS (Score = 2); A3, OS (Score = 6); RMS (Score = 7); B. IHC of CXCL6: B1, OS (Score = 7); B2, CS (Score = 4); B3, OS (Score = 7); B4, RMS (Score = 7); C. IHC of CXCL12: C1, OS (Score = 5); C2, CS (Score = 0); C3, OS (Score = 4); C4, RMS (Score = 0).

Table 4.

Immunohistochemistry of the three chemokines in two OS tissue arrays.

A. Summary of the IHC scores in the immunohistochemistry results.
Protein IHC score Biomax Tissue Array
 COG Tissue Array
OS (%) CS (%) OS (%) RMS (%)
CXCL4 0 0 (0%) 11 (39%) 0 (0%) 0 (0%)
1–4 9 (18%) 10 (36%) 8 (12%) 0 (0%)
5–7 40 (82%) 7 (25%) 56 (88%) 10 (100%)
CXCL6 0 10 (20%) 19 (68%) 0 (0%) 0 (0%)
1–4 22 (45%) 5 (18%) 12 (18%) 1 (10%)
5–7 17 (35%) 4 (14%) 52 (82%) 9 (90%)
CXCL12 0 46 (94%) 28 (100%) 63 (98%) 10 (100%)
1–4 0 (0%) 0 (0%) 1 (2%) 0 (0%)
5–7 3 (6%) 0 (0%) 0 (0%) 0 (0%)
B. The intensity score and proportion score of positive cells in the tumors with high IHC scores of CXCL4.
IHC Score
 Biomax Tissue Array
 COG Tissue Array
Total Intensity Score Proportion Score OS (%) CS (%) OS (%) RMS (%)
5 1 4 1 (2%) 1 (3%) 11 (17%) 1 (10%)
2 3 15 (31%) 3 (11%) 10 (16%) 1 (10%)
3 2 0 (0%) 2 (7%) 0 (0%) 0 (0%)
6 2 4 10 (20%) 0 (0%) 30 (47%) 6 (60%)
3 3 1 (2%) 0 (0%) 0 (0%) 1 (10%)
7 3 4 13 (27%) 1 (3%) 5 (8%) 1 (10%)
# of High IHC Score Cases (%) 40 (82%) 7 (25%) 56 (88%) 10 (100%)
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The numbers in the table represent the total number of tumor cores that had the specific IHC scores. IHC score: 0, no evidence of expression; 1–4, weak expression; 5–7, strong expression.

The description of the scores is listed in the Materials and Methods.

The Three Chemokines Promotes Proliferation of OS Cells in vitro

We further tested if the three chemokines could stimulate the proliferation of four commonly used OS cell lines (U2-OS, SaOS-2, MG-63, SJSA). The results showed that all three chemokines significantly promoted the cell proliferation in all four OS cell lines (p < 0.05, Fig. 3). These results suggest that a high level of these chemokines in the local tumor environment or circulation may promote the tumor growth in the patients.

Fig. 3.

Fig. 3

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Exogenous CXCL4, CXCL6, and CXCL12 significantly stimulated the proliferation of four OS cell lines (A, U2-OS; B, SaOS-2; C, MG63; and D, SJSA) under a serum-deprived condition (* denotes p < 0.05). Y-axis is the optical density (OD).

CXCL4 and CXCL6 were Associated with the Patient Outcome

Our expression and functional results argue that these chemokines may play a role in the prognosis of OS patients. We, therefore, correlated the plasma levels of these three chemokines with the overall survival of the OS patients in the discovery set, where we had clinical follow-up information. We found that the higher levels of both CXCL4 (p = 0.007) and CXCL6 (p = 0.03), but not CXCL12 (p = 0.282), were significantly associated with a poor outcome (Fig. 4). These results were consistent with our findings that only CXCL4 and CXCL6 are expressed frequently and strongly in OS tissues. We then tested if the correlation of the poor outcome was due to an association with the metastatic disease at diagnosis in these patients. However, none of the three chemokines significantly correlated with metastasis.

Fig. 4.

Fig. 4

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Survival analysis of the three chemokines in OS patients. Low and high denote low and high chemokine levels, respectively, in the patients. Higher levels of CXCL4 (A) and CXCL6 (B), but not CXCL12, were significantly associated with a poor outcome (log-rank test, p < 0.05).

DISCUSSION

In this study, we identified 20 proteins that were differentially abundant in the OS plasma samples. Some of these proteins have been reported to correlate with cancer, such as TGF-beta RIII18 and Smad419. Among the eight elevated proteins in OS, three of them (CXCL4, CXCL6, and CXCL12) belong to the CXC chemokine family. Using ELISA, we validated that the higher levels of the three chemokines were associated with OS in the independent test set. These findings suggest that these three CXC chemokines may be useful for early diagnosis of OS. This is particularly important for children with a known predisposition to OS, such as patients harboring germline mutations in the TP5320, RB21, or Rothmund-Thomson Syndrome patients with RECQL5 mutations22. A blood-based assay of high-risk individuals may allow more timely and periodic monitoring of the disease and save lives. However, further studies of the circulating chemokines in patients with other sarcomas or bone diseases will be needed to test if the chemokines are specific to OS.

Previous studies have reported that CXCL4 is elevated in a variety of gynecologic malignancies23,24 and prostate cancer25; however, its role in OS is largely unknown. The primary function of CXCL4 is involved in platelet activation26. In our study, CXCL4 was consistently increased in the OS patients of both discovery and validation sets (60-fold higher). The serum samples used in the validation phase suggest that the higher level of this chemokines in OS patients was not due to platelet contamination of the plasma samples. In fact, the elevation of CXCL4 is consistent with the enhanced platelet activation observed in cancer patients that contributes to the increased occurrence of thrombo-embolic events27. Increased platelets may also provide protection of metastatic cells in the circulation28,29.

Interestingly, CXCL6 was first discovered from the culture medium of the OS cell line MG-63 after stimulation with IL-1β30. Our results showed that CXCL6 was expressed in OS. However, a strong CXCL6 expression was observed in a higher proportion of OS samples in the COG array (82%) than those from the Biomax array (35%). This may reflect the heterogeneity of the OS cases used in different arrays or the expression of CXCL6 in OS is inherently more variable. We have also observed that the tumor expression patterns of CXCL4 and CXCL6 share some similarities. The expressions of the two chemokines in the OS are moderately correlated (r=0.37 in the COG array and r=0.56 in the Biomax array), which is consistent with the fact that both CXCL4 and CXCL6 genes are located at the 4q12-q13, while CXCL12 is located at 10q11.131.

CXCL12 is secreted by stromal cells from a variety of tissues such as bone marrow, lung, and liver32. The chemotactic effect of CXCL12 is mediated by interaction with its receptor CXCR4. The involvement of the CXCR4/CXCL12 axis has been implicated in tumor progression of a variety of cancers and cancer stem cells 33,34. In OS, the migration of OS cells expressing CXCR4 receptor follows CXCL12 gradient and the adhesion OS cells to endothelial and bone marrow stromal cells is promoted by a CXCL12 treatment. Using mouse models, CXCR4/CXCL12 axis was proved to be involved in the metastatic process of OS cells, as development of lung metastasis was significantly decreased in the OS mouse models by the administration of a CXCR4 inhibitor or antagonist 14,35. An earlier study has found that CXCR4 is highly expressed in metastatic OS 36 and lung expresses CXCL1237. These findings are consistent with our results that most of OS cases did not express CXCL12. The increased level of CXCL12 in the peripheral blood of OS patients may be derived from other host tissues, such as lung or metastasized OS cells.

Our results suggest that these chemokines may play a direct functional role in OS tumorigenesis. Platelet CXCL4 has been found to be elevated in the early tumor growth of liposarcoma, breast cancer, and OS38. CXCL6 can induce expression of PCNA in small cell lung cancer cell lines in vitro39, and melanoma cell growth in vivo40. In addition, CXCL12 induces the proliferation in some tumor cell lines, such as ovarian carcinoma41 and non-small cell lung cancer42. To test if the CXC chemokines had a general proliferative effect on tumor cells, we repeated the proliferation assay using three different types of non-OS cell lines available in our laboratory. They were TC7 (Ewing), HepG (liver hepatocellular carcinoma), and DAOY (medulloblastoma). Our results showed that the three chemokines significantly increased the proliferation of TC7 cells only but had no effects on the HepG and DAOY cells (data not shown). These results suggest that the three CXC chemokines only promote the growth for certain types of tumors. A recent study has shown that many chemokine receptors are expressed in OS, such as CXCR3, CXCR4 and CXCR543. However, CXCR1, the cognate receptor of CXCL6 was not reported. The receptor for CXCL4 in the tumor cell is still not clear. Peptide-based or small molecules that can inhibit the functions of the chemokines or receptors may provide an alternative and effective therapy for OS patients who have a higher level of these chemokines44. For instance, inhibitor molecules of CXCR4, the receptor of CXCL12, have been already used in four clinical trials for various cancers (ClinicalTrials.gov database). Therefore, the development of inhibitors for other chemokines in OS is feasible. Since there is no targeted therapy for OS other than chemotherapy, our findings provide a foundation for future studies of these chemokines and their receptors, which may lead to a novel therapeutic approach to stop tumor growth in OS.

Our results showed that both CXCL4 and CXCL6 significantly correlated with patient outcomes; however, no associations between these two chemokines and the metastasis status at diagnosis were found. The high expression of these two chemokines in the tumor and plasma of OS patients may exert a strong proliferative effect and promote tumor progression and, hence, a poorer outcome of the patients. In contrast, plasma CXCL12 is likely to be expressed by other parts of the body and the difference between the plasma levels of the protein in OS patients and healthy controls was relative small when compared to the other two chemokines (Table 3); thus, the plasma level of CXCL12 did not significantly correlate with the patient outcomes. Despite the promising results in this study, we understand that our results were based on a relatively small group of patients and thus, they need to be interpreted with caution. A larger patient cohort is needed to validate the use of CXCL4 and CXCL6 as prognostic factors in OS. We have collaborated with the Children’s Oncology Group to use the samples collected from the European and North American OS Study to validate these findings.

Acknowledgments

We would like to thank William Meyer at University of Oklahoma Health Sciences Center for contributing osteosarcoma plasma for this study. We are also grateful to Carolyn Pena for assisting with the manuscript’s preparation. We thank Mark Krailo, Don Barkauskas, Susan Conway, and Chand Khanna from the Children’s Oncology Group for their help in retrieving clinical information of the osteosarcoma cases.

Grant Support: The work is supported by funding from the National Cancer Institute (1U01CA088126-01 and 1U01CA114757-01) and the Gillson Longenbaugh Foundation (CL), as well as the Wendy Will Case Cancer Foundation, the Sarcoma Foundation of America, and the Fleming and Davenport Award of Texas Medical Center (TKM). This research is also supported by the Chair's Grant U10 CA98543 and the Human Specimen Banking Grant U24 CA114766 of the Children's Oncology Group from the National Cancer Institute, National Institutes of Health, Bethesda, MD, USA. Additional support for this research is provided by an endowment from the WWWW (QuadW) Foundation, Inc. to the Children's Oncology Group.

Abbreviations

OS

Osteosarcoma

RMS

Rhabdomyosarcoma

CS

Chondrosarcoma

D-CTL

Disease control

N-CTL

Healthy control

Footnotes

Conflict of Interest Disclosures

The authors made no disclosures.

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