Abstract
Background: Increasing evidence has shown that Toll-like receptors (TLRs), key receptors in innate immunity, play a role in cancer development and progression. The present study aimed to elucidate the role of TLR expression in osteosarcoma cancer cells and patient specimens. Materials and Methods: We investigated the expression of all of human TLRs in osteosarcoma MG-63 cells by real-time quantitative reverse transcription polymerase chain reaction. We then further explored whether the up-regulation of TLR9 expression is common in patients with osteosarcoma by examining TLR9 protein levels in 80 osteosarcoma specimens and 28 normal controls by immunohistochemistry. Results: We found that among TLR family members, TLR9 was predominately expressed in osteosarcoma cells, and up-regulation of TLR9 expression was found in 72 out of 80 (90%) patients with osteosarcoma but in none of 28 normal controls. Furthermore, high expression of TLR9 appeared to be associated with osteosarcoma progression. Conclusion: TLR9 is up-regulated in the majority of osteosarcomas, which appears to play an important role in osteosarcoma development and progression. Therefore, TLR9 may serve as a novel therapeutic target for human osteosarcoma therapy.
Osteosarcoma, an aggressive and highly malignant cancer, is the most common bone tumor in children and adolescents (1). In about 15% to 20% of patients, osteosarcoma has metastasized by the time it is diagnosed (2). Once metastasis occurs, the mortality rate of patients dramatically increases, up to more than 80%, no matter how they are treated (1, 2). The cause for this disease is obscure. Multiple factors play a role in the disease development, including those related to genetics, epigenetics, environment, as well as microenvironment and immune responses, in which the interaction between cancer cells and immune cells may play a crucial role (1). The cross-talk between cancer cells and immune cells may also affect the treatment and prognosis of patients with the disease. Therefore, a better understanding of the role of factors in the immune system, such as Toll-like receptors, may provide a novel strategy to better control osteosarcoma.
Toll-like receptors (TLRs) are membrane-bound receptors that play key roles in both the innate and adaptive immune systems, particularly in inflammatory responses against pathogenic infection (3-5). These receptors are primarily expressed on innate immune cells and recognize conserved pathogen-associated molecular patterns (6). TLRs can also recognize endogenous ligands called danger-associated molecular pattern molecules (DAMPs) (7). Cancer cells may release many DAMPs at different stages. Activation of TLRs may play a role in cancer progression and development (8-10); however, activation of different TLRs might display completely different patterns. TLRs generally share a common signaling pathway; all of the TLRs except TLR3 go through the myeloid differentiation primary response gene 88 (MyD88)-dependent pathway (11). The activation of the TLR signaling pathway induces the nuclear factor-kappa B and mitogen-activated protein kinases (MAPK) activation to ultimately induce the secretion of many cytokines and chemokines, as well as interferon (11, 12). All these effectors play an important role in cancer development. However, the activation of different TLRs most likely also contributes different roles in cancer. It has been shown that activation of TLR4 signaling by lipopolysaccharides protects tumor cells from immune attack and thus promotes tumor growth (9). Triggering of TLR9 on cancer cells has been shown to protect cancer cells against the tumor necrosis factors-related apoptosis-inducing ligand-induced apoptosis and promotes tumor cell proliferation (13). In contrast, activation of TLR3 induces an anti-proliferative signaling event in human breast cancer and melanoma cells (10, 14). Thus, the function and biological importance of TLRs expressed on various tumor cells appears complex.
The role of TLRs in osteosarcoma has yet to be well-defined. Herein, we first investigated the expression of all 10 human TLRs in the osteosarcoma cell line. We then confirmed the specific TLR that is highly expressed in specimens from patients with osteosarcoma, and further explored the association between the expression level of the highly expressed TLR and osteosarcoma progression.
Materials and Methods
Reagents and cell line. Monoclonal antibody against TLR9 was purchased from Abcam (Cambridge, MA, USA). MG-63 osteosarcoma cell line was purchased from the American Type Culture Collection (Manassas, VA, USA) and maintained in Dulbecco's modified Eagle's medium, supplemented with 10% fetal bovine serum (GE Healthcare Life Sciences, HyClone Laboratories, Logan, UT, USA) and penicillin-streptomycin (Life Technologies, Grand Island, NY, USA). Human osteosarcoma tissue arrays (Table I and II), including 80 individual osteosarcoma specimens and 28 normal controls were purchased from US Biomax (Rockville, MD USA).
Real-time quantitative reverse transcription PCR (qRT-PCR). Real-time qRT-PCR was performed as previously described (15). Briefly, total RNA was isolated from cells using the RNAeasy kit (Omega Bio-Tek, Norcross, GA, USA). One microgram of total RNA was reverse transcribed using Superscript II reverse transcriptase (Invitrogen, Grand Island, NY, USA). The reverse transcription reaction was diluted 1:10, and 2 μl of the diluted sample was added to an 18-μl PCR assay mixture containing a 0.5 μM concentration of each primer and 1x SYBR Green JumpStart Taq ReadyMix (Sigma-Aldrich, St. Louis, MO, USA). PCR was conducted with the MyiQ single-color real time PCR detection system (Bio-Rad, Hercules, CA, USA) using the following conditions: hot start activation at 95°C for 10 min followed by 40 cycles of 95°C for 15 s, 61°C for 30 s, and 72°C for 30 s. Two sets of PCR assays were performed for each sample using the primers listed in Table III. The threshold cycle number for TLRs was normalized to that of β-actin, and the resulting value was converted to a linear scale. All assays were performed at least three times from independent RNA preparations.
Immunohistochemistry. Immunohistochemical analyses were performed with the ABC Elite Kit (Vector Laboratories, Burlingame, CA, USA) (8). Slides were deparaffinized first, and then washed with the phosphate-buffered saline (PBS) and blocked with normal goat serum (1%) plus 1% bovine serum albumin (BSA) in PBS for 20 minutes at room temperature. Slides were then incubated overnight with primary antibodies (1:100) against TLR9 in 1x PBS with 1% BSA. Samples were washed with PBS and incubated with biotin anti-rabbit secondary antibody (1:200) for 1 h. Next, samples were washed with PBS and incubated with streptavidin–horseradish peroxidase conjugate solution for 20 min. After washing with PBS, the slides were then mounted with mounting solution. The TLR9 expression level in all the sections was evaluated by two independent investigators in a double-blind manner, and staining intensity was graded as negative and positive (including weak and strong).
Toll-like receptor 9 expression in osteosarcoma and normal controls.
Statistical analysis. p-Values for differences in expression were calculated with Fisher's exact test using PRISM, version 5 (GraphPad software, La Jolla, CA USA).
Results
Osteosarcoma cells predominantly expressed TLR9. We found that TLRs, key pattern recognition receptors in the innate immune system, are expressed not only in innate immune cells, such as macrophages and dendritic cells but also in cancer cells such as breast cancer cells (8) and prostate cancer cells (16). Herein, we wanted to determine whether TLRs are also expressed in osteosarcoma cells. By utilizing real-time quantitative RT-PCR, we investigated the expression profile of all human TLRs in MG-63 osteosarcoma cells. Surprisingly, among TLR family, TLR9 is predominantly expressed in osteosarcoma MG-63 cells, although TLR2 and TLR4 are also expressed in osteosarcoma cells (Figure 1).
TLR9 is up-regulated in human osteosarcoma and appears to be associated with disease progression. The finding that TLR9 is highly up-regulated in osteosarcoma MG-63 cells led us to investigate whether TLR9 is up-regulated in patients with osteosarcoma and whether TLR9 expression is associated with osteosarcoma progression. We then performed immunohistochemical staining with TLR9-specific antibody to investigate TLR9 expression in 108 human specimens on two tissue arrays from US Biomax, including 28 normal bone tissues and 80 osteosarcoma specimens with different sub-types, stages, and grades. We found that no normal bone tissues expressed TLR9 (Figure 2). However, among the 80 osteosarcoma specimens, 72 (90%) were positive for TLR9 expression (Table I). Intriguingly, the expression level of TLR9 protein in the high-grade osteosarcomas appeared to be higher than in the low-grade tumors (Figure 2), although more specimens in different stages are needed to draw a definite conclusion. Furthermore, the positive staining appeared to be localized in the cytoplasm, which corresponded with the concept of TLR9 as one of intracellular TLRs. This finding indicates that up-regulation of TLR9 plays a role in osteosarcoma development and the TLR9 expression level may be associated with human osteosarcoma progression (Table II).
Summary of clinical significance of Toll-like receptor 9 expression in osteosarcoma.
Discussion
In the present study, we investigated the expression of all of human TLRs in osteosarcoma cells and identified that among TLR family members, TLR9 is predominately expressed in osteosarcoma cells. We further explored whether this up-regulation of TLR9 expression is common in osteosarcoma patients by determining TLR9 protein level in 80 osteosarcoma specimens and 28 normal controls. Indeed, we found TLR9 up-regulation in 90% of osteosarcoma patient specimens, but none of the normal controls showed TLR9 expression. More importantly, the TLR9 expression level appears to be associated with osteosarcoma progression, although this association needs to be further confirmed with a larger sample size.
The discovery that TLR9 is predominately up-regulated among TLR family members in osteosarcoma is surprising and intriguing. We, as well as others, have found that different cells of different cancer types, such as breast cancer (8) and prostate cancer (16), generally express multiple, different TLRs (17, 18). As for an uncommon characteristic that one type of cancer cell predominately expressed one TLR, our findings may indicate that, specifically, TLR9 might play an important role in osteosarcoma development.
The expression profile of toll-like receptors (TLR) in human osteosarcoma MG-63 cells. Osteosarcoma cells predominantly expressed TLR9, as determined by real-time quantitative reverse transcription polymerase chain reaction.
The mechanism underlying TLR9 up-regulation in osteosarcoma is not distinctly understood, and the cause of the majority of osteosarcomas is not clear at the time. TLRs are well-known for their recognition of pathogen-associate molecular patterns in infection, but the pathogens, including bacteria, viruses, fungi and parasites, are rarely associated with the risk of osteosarcoma. However, recent findings show that certain changes in DNA can cause cells to become cancerous (19, 20), which most likely serves as a mechanism for osteosarcoma development. These discoveries aid in establishing a perfect link between osteosarcoma and TLR9, which generally recognizes microbial CpG DNA, as well as endogenous damaged DNA (21).
TLR9 is an intracellular TLR and resides predominately in the endoplasmic reticulum in resting cells. TLR9 is primarily expressed in plasmacytoid dendritic cells and B-cells (22), and recognizes unmethylated CpG DNA (23), which is a component of bacterial genomes, and viral DNA but is rarely present in vertebrate genomes. Activation of TLR9 in plasmacytoid dendritic cells and B-cells results in potent T-helper-1-type immune responses and antitumor activity. Upon binding to its ligand in the endosome, TLR9 signaling is activated by recruitment of MyD88 to induce the secretion of pro-inflammatory cytokines and type-I interferon (24).
Tumor immunotherapy has become an attractive and hot research area. One of the earliest successes in immunotherapy utilized attenuated in mycobacterium Bacillus Calmette-Guerin (BCG) to treat bladder cancer. The active ingredient in BCG is actually DNA, which induces an immune response and tumor regression (25, 26).
Toll-like receptor 9 (TLR9) expression in osteosarcoma. Human osteosarcoma tissue arrays were subjected to immunohistochemistry analyses with monoclonal primary antibody to TLR9 and biotinylated second antibody. Brown indicates positive staining for TLR9. Representative images from tissues with different histological type are shown: no TLR9 expression in normal bone tissue; moderate TLR9 expression in osteosarcoma stage IA; strong TLR9 expression in osteosarcoma stage IIB. Magnification, ×40.
Quantitative PCR primers for human Toll-like receptors.
TLR9 agonists and their derivatives have been evaluated in various clinical trials with promising therapeutics in the treatment of certain types of cancer, including melanoma, T-cell lymphoma, kidney cancer and lung cancer (13); however, the limitation for further utilization of these TLR agonists is their severe side-effects if we are not able to control the administration to specific cells in a timely manner. In terms of therapeutic agents, in order to increase efficiency, TLR9 agonists have been commonly combined with other anti-tumor strategies. However, other factors could alter the efficiency of TLR9 stimulation of the generation of an antitumor response. In osteosarcoma, considering TLR9 is predominately expressed in cancer cells, CpG oligodeoxynucleotide (ODN) monotherapy might be sufficient to induce tumor regression and is worth further investigation.
In conclusion, we found that among TLR family members, TLR9 is predominately expressed in osteosarcoma cells. This up-regulation of TLR9 expression is common in patients with osteosarcoma, being present in 90% of osteosarcomas but absent in normal controls. TLR9 expression level appears to be associated with osteosarcoma progression. Therefore, TLR9 may serve as a novel therapeutic target for human osteosarcoma therapy.
Acknowledgements
This study was supported in part by NIH grant CA176698 (D.Z.).
Footnotes
Conflicts of Interest
No potential conflicts of interest were disclosed.
- Received July 14, 2015.
- Revision received September 11, 2015.
- Accepted September 16, 2015.
- Copyright© 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved
