Abstract

Introduction

Bone is a very dynamic tissue resulting from coordinated phases of formation and resorption called bone remodeling. In addition to its role in phosphocalcic homoeostasis, the bone remodeling process is necessary for bone growth and renewal of cellular and extracellular matrix components to adapt bone organization to the various biological and mechanical constraints (¹1. Dempster DW. Anatomy and functions of the adult skeleton. In: Favus MJ (Editor). Primer of the metabolic bone diseases and disorders of mineral metabolism. 6th edn. Durham: American Society for Bone and Mineral Research Publication Office; 2006. p 7–11. 2. Favus MJ, Bushinsky DA, Lemann J Jr. Regulation of calcium, magnesium, and phosphate metabolism. In: Favus MJ (Editor). Primer of the metabolic bone diseases and disorders of mineral metabolism. 6th edn. Durham: American Society for Bone and Mineral Research Publication Office; 2006. p 76–83.–³3. Deschaseaux F, Sensebe L, Heymann D. Mechanisms of bone repair and regeneration. Trends Mol Med 2009; 15: 417–429, doi: 10.1016/j.molmed.2009.07.002.
https://doi.org/10.1016/j.molmed.2009.07... ). Multiple myeloma also has a strong preference for growth and metastasis in the bone marrow. The most common sites of bone metastases are the spine, ribs, pelvis, proximal femur, and skull. Breast cancer preferentially metastasizes to the lungs and bones, whereas prostate cancer almost exclusively metastasizes to the bones (⁴4. Hess KR, Varadhachary GR, Taylor SH, Wei W, Raber MN, Lenzi R, et al. Metastatic patterns in adenocarcinoma. Cancer 2006; 106: 1624–1633, doi: 10.1002/cncr.21778.
https://doi.org/10.1002/cncr.21778... ).

Bone metastases can be classified as osteolytic with significant bone destruction, osteoplastic due to excess bone formation, or a mixed phenotype can occur (⁵5. Kraljevic Pavelic S, Sedic M, Bosnjak H, Spaventi S, Pavelic K. Metastasis: new perspectives on an old problem. Mol Cancer 2011; 10: 22, doi: 10.1186/1476-4598-10-22.
https://doi.org/10.1186/1476-4598-10-22... ). Metastasis to the bones is facilitated by the fenestrated structure of the bone marrow sinusoid capillaries, high blood flow in the areas of red marrow, and adhesive molecules on tumor cells that bind to the bone marrow stromal cells such as osteoblasts and osteoclasts as well as the bone matrix (⁶6. Nguyen DX, Bos PD, Massaggue J. Metastasis: from dissemination to organ-specific colonization. Nat Rev Cancer 2009; 9: 274–284, doi: 10.1038/nrc2622.
https://doi.org/10.1038/nrc2622... ). These cell types play an important role in bone remodeling and niche structure (⁷7. Saki N, Abroun S, Farshdousti Hagh M, Asgharei F. Neoplastic bone marrow niche: hematopoietic and mesenchymal stem cells. Cell J 2011; 13: 131–136.). The bone marrow microenvironment includes osteoblastic (endosteal) and vascular niches, which provide an environment that supports hematopoietic and non-hematopoietic stem cells (⁸8. Azizidoost S, Babashah S, Rahim F, Shahjahani M, Saki N. Bone marrow neoplastic niche in leukemia. Hematology 2014; 19: 232–238, doi: 10.1179/1607845413Y.0000000111.
https://doi.org/10.1179/1607845413Y.0000... ).

Bone homeostasis is maintained by balanced production of osteoblasts and osteoclasts. Disruption of this balance can convert normal niches into metastatic niches. Disseminated tumor cells in the bone marrow can persist in a dormant state for many years until they progress to a macrometastatic lesion (⁹9. Giancotti FG. Mechanisms governing metastatic dormancy and reactivation. Cell 2013; 155: 750–764, doi: 10.1016/j.cell.2013.10.029.
https://doi.org/10.1016/j.cell.2013.10.0... ). The clinical challenges associated with bone metastasis are deleterious effects such as bone pain, fractures, life-threatening hypercalcemia, spinal cord compression, and other nerve compression syndromes (¹⁰10. Theriault RL, Theriault RL. Biology of bone metastasis. Cancer Control. 19: 92–101.). Deeper understanding of the process of bone metastasis and identification of targets for prevention and treatment is, therefore, an objective of paramount importance.

Osteoblasts and osteoclasts are crucial for interactions with tumor cells in the context of their colonization of the bone marrow. Osteoclasts are multinucleated cells that arise from precursor cells of the monocyte-macrophage lineage. They de-mineralize bone by dissolving calcium phosphate crystals and producing acid and are involved in bone resorption through degradation of the newly-exposed extracellular matrix (ECM) by secreted proteases. Osteoclasts adhere to bone surface and the spectrum of factors involved in their activation may depend on tumor type. Osteoblasts are specialized, terminally differentiated mononuclear cells differentiated from mesenchymal stem cells and are involved in bone reconstruction by generation of new ECM and deposition of calcium phosphate crystals into the interstices of the matrix (¹¹11. Mundy GR. Metastasis to bone: causes, consequences and therapeutic opportunities. Nat Rev Cancer 2002; 2: 584–593, doi: 10.1038/nrc867.
https://doi.org/10.1038/nrc867... ). Prerequisite for the generation of osteolytic metastases is the activation of osteoclasts. To this end, osteoblasts secrete receptor activator of NF-κB ligand (RANKL), which interacts with osteoclast precursors displaying RANK receptor on their surface, resulting in their activation and finally maturation into functional osteoclasts.

Osteoblasts also produce osteoprotegerin (OPG), a soluble decoy receptor which can block RANK/RANKL signaling by scavenging of RANKL. Thus, the activation of osteoclasts is triggered by the balance between RANKL and OPG (¹²12. Dougall WC. Molecular pathways: osteoclast-dependent and osteoclast-independent roles of the RANKL/RANK/OPG pathway in tumorigenesis and metastasis. Clin Cancer Res 2012; 18: 326–335, doi: 10.1158/1078-0432.CCR-10-2507.
https://doi.org/10.1158/1078-0432.CCR-10... –¹³13. Dougall WC, Holen I, Gonzalez Suarez E. Targeting RANKL in metastasis. Bonekey Rep 2014; 3: 519, doi: 10.1038/bonekey.2014.14.
https://doi.org/10.1038/bonekey.2014.14... ). RANKL also can induce factors involved in migration, invasion, and angiogenesis such as matrix metalloproteinases 1 and 9 (MMP1, MMP9), matrix metalloproteinase inducer EMMPRIN/CD147, intercellular adhesion molecule-1, interleukin 6 and 8 (IL6, IL8), and vascular endothelial growth factor (¹⁴14. Rucci N, Millimaggi D, Mari M, Del Fattore A, Bologna M, Teti A, et al. Receptor activator of NF-kappaB ligand enhances breast cancer-induced osteolytic lesions through upregulation of extracellular matrix metalloproteinase nducer/CD147. Cancer Res 2010; 70: 6150–6160, doi: 10.1158/0008-5472.CAN-09-2758.
https://doi.org/10.1158/0008-5472.CAN-09... ) and decrease the expression of metastasis suppressor serpin 5b/maspin (¹⁵15. Luo JL, Tan W, Ricono JM, Korchynskyi O, Zhang M, Gonias SL, et al. Nuclear cytokine-activated IKKα controls prostate cancer metastasis by repressing maspin. Nature 2007; 446: 690–694, doi: 10.1038/nature05656.
https://doi.org/10.1038/nature05656... ). RANKL can also promote the function of regulatory T-cells and macrophages (¹⁶16. Ferrari-Lacraz S, Ferrari S, Do RANKL inhibitors (denosumab) affect inflammation and immunity? Osteoporos Int 2007; 22: 435–446, doi: 10.1007/s00198-010-1326-y.
https://doi.org/10.1007/s00198-010-1326-... ).

Osteolysis is based on a self-perpetuating signaling system (vicious cycle) that is maintained by mitogenic factors for tumor cells such as transforming growth factor-β, insulin-like growth factor-1, fibroblast growth factors, platelet derived growth factors, and Ca-ions released from demineralized bone as well as parathyroid hormone-related peptide (PTHrP) derived from tumor cells. PTHrP acts as a promotor of osteolysis by osteoclasts (¹⁷17. Soki FN, Park SI, McCauley LK. The multifaceted actions of PTHrP in skeletal metastasis. Future Oncol 2012; 8: 803–817, doi: 10.2217/fon.12.76.
https://doi.org/10.2217/fon.12.76... ).

CD147, a transmembrane protein of the immunoglobulin (Ig) superfamily, was identified independently in different species and has many designations across different species such as M6, neurothelin, 5A11, HT7, OX-47, CE9, EMMPRIN, basigin, and gp42 (¹⁸18. Hanna SM, Kirk P, Holt OJ, Puklavec MJ, Brown MH, Barclay AN. A novel form of the membrane protein CD147 that contains an extra Ig-like domain and interacts homophilically. BMC Biochem 2003; 4: 17, doi: 10.1186/1471-2091-4-17.
https://doi.org/10.1186/1471-2091-4-17... 19. Fossum S, Mallett S, Barclay AN. The MRC OX-47 antigen is a member of the immunoglobulin superfamily with an unusual transmembrane sequence. Eur J Immunol 1991; 21: 671–679, doi: 10.1002/eji.1830210320.
https://doi.org/10.1002/eji.1830210320... –²⁰20. Seulberger H, Lottspeich F, Risau W. The inducible blood rain barrier specific molecule HT7 is a novel immunoglobulin like cell surface glycoprotein. EMBO J 1990; 9: 2151–2158, doi: 10.1002/j.1460-2075.1990.tb07384.x.
https://doi.org/10.1002/j.1460-2075.1990... ). Soluble CD147 also has been detected in microvesicles (exosomes) (²¹21. Millimaggi D, Mari M, D'Ascenzo S, Carosa E, Jannini EA, Zucker S, et al. Tumor vesicleassociated CD147 modulates the angiogenic capability of endothelial cells. Neoplasia 2007; 9: 349–357, doi: 10.1593/neo.07133.
https://doi.org/10.1593/neo.07133... –²²22. Simpson RJ, Jensen SS, Lim JW. Proteomic profiling of exosomes: current perspectives. Proteomics 2008; 8: 4083–4099, doi: 10.1002/pmic.200800109.
https://doi.org/10.1002/pmic.200800109... ). Homotypic interactions may play an important role regarding the mode of action of MMP induction since it has been shown that recombinant CD147 fusion proteins can interact homotypically (²³23. Sun J, Hemler ME. Regulation of MMP-1 and MMP-2 production through CD147/extracellular matrix metalloproteinase inducer interactions. Cancer Res 2001; 61: 2276–2281.). However, interaction of CD147 with an as yet unidentified receptor might also be responsible for the phenomena as described above.

CD147 can be induced in tumor cells by epidermal growth factor receptor mediated signaling (autocrine mechanism) (²⁴24. Menashi S, Serova M, Ma L, Vignot S, Mourah S, Calvo F. Regulation of extracellular matrix metalloproteinase inducer and matrix metalloproteinase expression by amphiregulin in transformed human breast epithelial cells. Cancer Res 2003; 63: 7575–7580.). Controlled degradation of the extracellular matrix is a prerequisite for tumor invasion and metastasis. Transfection of breast cancer cells with CD147 resulted in increased tumor growth and metastasis after implantation into the mammary gland and correlated with high levels of MMP-2 and MMP-9 (²⁵25. Zucker S, Hymowitz M, Rollo EE, Mann R, Conner CE, Cao J, et al. Tumorigenic potential of extracellular matrix metalloproteinase inducer. Am J Pathol 2001; 158: 1921–1928, doi: 10.1016/S0002-9440(10)64660-3.
https://doi.org/10.1016/S0002-9440(10)64... ). The molecular mechanisms of these phenomena have been poorly resolved. Glycosylated CD147 was shown to be a prerequisite for MMP induction at the transcriptional level (²⁵25. Zucker S, Hymowitz M, Rollo EE, Mann R, Conner CE, Cao J, et al. Tumorigenic potential of extracellular matrix metalloproteinase inducer. Am J Pathol 2001; 158: 1921–1928, doi: 10.1016/S0002-9440(10)64660-3.
https://doi.org/10.1016/S0002-9440(10)64... ). Induction of MMPs by CD147 was prevented with a monoclonal antibody directed against CD147.

In all bone cancers, a strong relationship between tumor cells and bone micro-environment has been clearly established, facilitating the tumor development and/or the metastatic process. These specific communication pathways have strongly stimulated research and development programs to design new drugs to treat oncologic bone diseases and have led specifically to the development of therapies targeting RANKL. The current study represents a comprehensive evaluation of CD147 expression pattern in bone cancer. The results of this study will provide the necessary background information for further research on the regulation and function of this intriguing peptide.

Material and Methods

Clinical samples

Malignant bone tumor and benign bone tumor tissue samples from 64 patients (mean age of 57.12±11.31 years, male:female 39:25) were collected at the time of dissection from patients at the Changhai Hospital from July 2014 to December 2016. The biopsies were frozen and stored at –80°C until preparation of total RNA. All tumor tissues were histologically confirmed to be from bone tumor. The study was approved by the ethics committee of Changhai Hospital, China.

Cell treatment

RAW264.7 cells were purchased from ATCC (USA) and maintained in RPMI 1640 with 10% (v/v) FBS (Invitrogen, USA). RAW264.7 cells in 6-well plates (2×10⁷ per well) in a humidified incubator with 5% CO₂ at 37°C were supplemented with RANKL (PeproTech, 100 ng/mL) and then transfected with pcDNA 3.1/CD147 or si-RANKL. All cells were divided into five groups with different treatments: Control: no treatment; CD147+RANKL: over-expression of CD147 and RANKL (100 ng/mL); RANKL: RANKL (100 ng/mL); CD147+si-RANKL: over-expression of CD147 and knock-down of RANKL; si-RANKL: knock-down of RANKL alone; and constructing CD147: the full length of CD147 was amplified and cloned into pcDNA3.1 vectors (Invitrogen). si-RANKL was used for further studies, which were synthesized by GenePharma (China). The sequence of si-RANKL is listed in Table 1. Lipofectamine 2000 (Invitrogen) was used to transfect oligonucleotides and constructs into cell lines according to the manufacturer's instructions. The infection efficiency was confirmed by qRT-PCR or western blot 48 h after transfection.

TRACP staining

Tartrate-resistant acid phosphatase (TRACP) staining was performed by a modification of a previously described method (²⁶26. Wu X, Li Z, Yang Z, Zheng C, Jing J, Chen Y, et al. Caffeic acid 3,4-dihydroxy-phenethyl ester suppresses receptor activator of NF-κB ligand-induced osteoclastogenesis and prevents ovariectomy-induced bone loss through inhibition of mitogen-activated protein kinase/activator protein 1 and Ca2+-nuclear fact. J Bone Miner Res 2012; 27:1298–1308, doi: 10.1002/jbmr.1576.
https://doi.org/10.1002/jbmr.1576... ) and the amount of TRACP positive multinucleated cells was calculated. Cells were fixed and stained for TRACP according to manufacturer instructions after 7 days of culture in the presence of RANKL or CD147.

Western blot analysis

Total cellular protein in RAW264.7 cells with different treatments was isolated by the addition of 1% PMSF and RIPA lysis buffer (50 mM Tris-HCl (pH 7.4) (Sigma-Aldrich, USA), 150m M NaCl, 1% NP-40, 0.1% SDS). After boiling with SDS-PAGE sample buffer for 5 min, the samples were prepared for sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Then, proteins were transferred onto a polyvinylidene difluoride membrane (Millipore, USA). After being blocked for 1 h at room temperature, the membrane was incubated with a 1:1000 dilution of rabbit polyclonal anti-mouse TRAF6, NFATc1, and β-actin antibody (ABGENT, USA) overnight. Before detected with an ECL chemiluminescence detection kit (Advansta, USA), proteins were incubated with the corresponding secondary antibody for 1 h at room temperature. The bands were obtained by GeneGnome 5 (Synoptics Ltd., UK).

RT-PCR

Total RNA was extracted from tissues using Trizol (Invitrogen). Reverse transcription of mRNA from malignant bone tumor, benign bone tumor, and RAW264.7 cells was carried out in 100 µL final volume from 400 ng total-RNA using high capacity cDNA Archive kit (Applied Biosystems, USA) according to manufacturer's instructions. The levels of mRNAs were measured by real-time quantitative RT-PCR (qRT-PCR) using Bio-Rad IQ5 system, and primers were utilized as described in Table 1. Reactions were performed in 50 µL volumes containing SYBR Green PCR master mix (Perkin-Elmer Biosystems, USA). Real-time PCR was performed using Bio-Rad IQ5 system in 96-well optical plates. Thermal cycling conditions were as follows: 2 min at 50°C and 10 min at 95°C, followed by 40 cycles at 95°C for 30 s, 60°C for 30 s, and 72°C for 2 min. Data were collected using the ABI analytical thermal cycler (Applied Biosystems). RNA expression was calculated based on a relative standard curve with ΔΔct method.

Statistical analysis

The measurement data are reported as means±SD. Independent continuous variables were compared using the t-test. P<0.05 was considered to be statistically significant. All calculations were made using SPSS 18.0 (SPSS Inc., USA).

Results

CD147 was up-regulated in malignant bone tumor

In order to investigate the conditions of CD147 expression in malignant bone tumor, CD147 cDNA was selected for further verification of differential expression by RT-PCR. Using specific primers designed for the CD147 cDNA clone (Table 1), RT-PCR was performed on malignant bone tumor and benign bone tumor tissues.

The result of this RT-PCR analysis is shown in Figure 1. CD147 mRNA was detected in malignant bone tumor and benign bone tumor tissues. CD147 expression levels were markedly up-regulated over half of the expression levels in the benign bone tumor tissues as well as the MMP-2 and MMP-9 expression levels. On the contrary, CD147 expression levels in the benign bone tumor tissues samples analyzed were lower than that in malignant bone tumor. Collectively, our results indicated that abnormal up-regulation of CD147 was confirmed in human malignant bone tumor.

Over-expression of CD147 can effectively promote osteoclast formation

Transfection was validated by RT-PCR that assessed increased ΔCt levels in transfected cells compared with Control and Blank (data not shown). We found that over-expressed RANKL progressively increased osteoclast formation up to 48 h of transfection (Figure 2), which suggested that RANKL can promote the formation of osteoclast. Up to 48 h, CD147+RANKL group had significantly increased processes of osteoclast formation compared with the other groups (P<0.05).

Knock-down of RANKL can effectively reduce the osteoclast formation compared with over-expression of RANKL. Therefore, it can be summarized that over-expression of CD147 and RANKL can promote the osteoclast formation.

Western blot of key factors in RANKL signaling pathway with over-expression of CD147

We evaluated the expressions of key factors such as TNF receptor-associated factor-6 (TRAF6) and nuclear factor of activated T-cells cytoplasmic 1 (NFATc1) using western blot (Figure 3) (²⁷27. Kim N, Kadono Y, Takami M, Lee J, Lee SH, Okada F, et al. Osteoclast differentiation independent of the TRANCE-RANK-TRAF6 axis. J Exp Med 2005; 202: 589–95, doi: 10.1084/jem.20050978.
https://doi.org/10.1084/jem.20050978... ). In the Control group (48 h), there was no significant difference in the protein expression of the two key factors. The expressions of TRAF6 in si-RANKL and CD147+si-RANKL group were down-regulated compared with control groups. Meanwhile, the expressions of NFATc1 in si-RANKL and CD147+si-RANKL groups were also down-regulated compared with control groups. However, the expression of NFATc1 in CD147+ RANKL group was significantly up-regulated compared with Control group. The results mentioned above suggested that over-expressed CD147 and RANKL genes can retain the expression of key factors in RANKL signaling pathway (in vitro).

RT-PCR analysis of osteoclast-specific expression genes with different treatments

The expressions of four osteoclast-specific expression genes, including TRACP, MMP-2, MMP-9, and c-Src, were analyzed by using RT-PCR, in which GAPDH was employed as a control for both cDNA quality and efficiency of the PCR amplification.

As shown in Figure 4, four osteoclast-specific expression genes were detectable in all osteoclasts with different treatments. However, the highest expression level of these genes appeared in the CD147+ RANKL group and the lowest appeared in the si-RANKL treatment.

Discussion

CD147 is a member of the immunoglobulin family of receptors. Members of this family play a role in intercellular communication involved in many immune-related functions, differentiation, and development. CD147 plays a role in spermatogenesis, lymphocyte activation, and expression of monocarboxylate transporters, and has been identified as a regulatory subunit of the γ-secretase complex in Alzheimer's disease amyloid β-peptide production (²⁸28. Yang D, Liu R, Liu L, Liao H, Wang C, Cao Z. Involvement of CD147 in alveolar bone remodeling and soft tissue degradation in experimental periodontitis. J Periodontal Res 2017; 52: 704–712, doi: 10.1111/jre.12435.
https://doi.org/10.1111/jre.12435... 29. Iacono KT, Brown AL, Greene MI, Saouaf SJ. CD147 immunoglobulin superfamily receptor function and role in pathology. Exp Mol Pathol 2007; 83: 283–295, doi: 10.1016/j.yexmp.2007.08.014.
https://doi.org/10.1016/j.yexmp.2007.08.... –³⁰30. Yuan S, Wang L, Chen X, Fan B, Yuan Q, Zhang H, et al. Triptolide inhibits the migration and invasion of human prostate cancer cells via Caveolin-1/CD147/MMPs pathway. Biomed Pharmacother 2016; 84:1776–1782, doi: 10.1016/j.biopha.2016.10.104.
https://doi.org/10.1016/j.biopha.2016.10... ). Some of these insights were obtained from the study of cd147-/- mice. These animals are defective in MMP regulation, spermatogenesis, lymphocyte responsiveness, and neurological functions at the early stages of development. Such female mice are infertile due to failure of implantation and fertilization (³¹31. Muramatsu T, Miyauchi T. Basigin (CD147): a multifunctional transmembrane protein involved in reproduction, neural function, inflammation and tumor invasion. Histol Histopathol 2003; 18: 981–987.). CD147 is involved in the transport of the MCT-1 and MCT-3 to the plasma membrane since reduced accumulation of these transporters has been observed in the retina of cd147 knockout mice.

A functional role of CD147 in cell adhesion is supported by its involvement in the blood-brain barrier and its interactions with integrins. CD147 has been implicated in many pathological processes, such as rheumatoid arthritis, experimental lung injury, atherosclerosis, chronic liver disease induced by hepatitis C virus, ischemic myocardial injury, and heart failure (³²32. Gabison EE, Hoang-Xuan T, Mauviel A, Menashi S. EMMPRIN/CD147, an MMP modulator in cancer, development and tissue repair. Biochimie 2005; 87: 361–368, doi: 10.1016/j.biochi.2004.09.023.
https://doi.org/10.1016/j.biochi.2004.09... ). Treatment of transplant patients with a CD147 antibody was effective due to inhibition of T-cell activation (³³33. Deeg HJ, Blazar BR, Bolwell BJ, Long GD, Schuening F, Cunningham J, et al. Treatment of steroid-refractory acute graft-versus-host disease with anti-CD147 monoclonal antibody ABX-CBL. Blood 2001; 98: 2052–2058, doi: 10.1182/blood.V98.7.2052.
https://doi.org/10.1182/blood.V98.7.2052... ). From this study, RT-PCR analysis showed that CD147 mRNA was detected in malignant bone tumor and benign bone tumor tissues. CD147 expression levels were markedly up-regulated over half of the expression levels in the benign bone tumor tissues.

A high incidence of CD147 expression in different cancer entities through tissue microarrays and monoclonal antibodies (mAb) MEM-M6/1 and HIM6 was noted in a systematic investigation (³⁴34. Riethdorf S, Reimers N, Assmann V, Kornfeld JW, Terracciano L, Sauter G, et al. High incidence of EMMPRIN expression in human tumors. Int J Cancer 2006; 119: 1800–1810, doi: 10.1002/ijc.22062.
https://doi.org/10.1002/ijc.22062... ). Several of the 2348 and 608 tissue samples covering 129 tumor types and 76 normal tissues, respectively, were investigated for their CD147 status with these antibodies. CD147 expression was found in 112 out of 129 tumor entities with the following incidences: squamous cell carcinomas (60–100%), pancreatic cancer (87%), chromophobic kidney cancer (83%), hepatocellular carcinoma (83%), medullary breast cancer (83%), and glioblastoma multiforme (79%).

A homogeneous expression of CD147 was found in tumors such as squamous cell carcinoma of different organs and mesotheliomas. The following normal tissues scored positively for CD147 expression: proliferatively active and differentiating epithelial cells, myocardial cells in the left heart ventricle, and vascular endothelial cells of the brain. Interestingly, CD147 isoforms differing in presence or absence of Lewis X glycan structures were found on breast cancer cells. Another investigation of expression and function of CD147 as a cancer-associated biomarker made use of mAb HAb18 (IgG1) (³⁵35. Li Y, Xu J, Chen L, Zhong WD, Zhang Z, Mi L, et al. HAb18G (CD147), a cancer-associated biomarker and its role in cancer detection. Histopathology 2009; 54: 677–687, doi: 10.1111/j.1365-2559.2009.03280.x.
https://doi.org/10.1111/j.1365-2559.2009... ). Several of the 28 tissue microarrays and 1117 pathological sections of breast tissue samples were analyzed. The incidence of CD147 expression was: cancer of the liver 80% (n=20), lung 62% (n=90), stomach 66% (n=44), colon 58% (n=19), rectum 59% (n=17), breast 64% (n=1055), brain 90% (n=52), esophagus 87% (n=16), ovary 75% (n=40), urinary bladder 85% (n=41), skin (squamous cell carcinoma) 58% (n=41), larynx 85% (n=63), and kidney 73% (n=33), and 30% of sarcomas such as osteo, chondro- and fibrosarcoma (n=102). Staining was ranked as weak, moderate, and strong. Strong staining was observed in 20% of breast, ovarian, and brain tumors. The level of CD147 expression was correlated with survival of the patients in a retrospective study of 106 patients with infiltrating ductal carcinoma of the breast (³⁵35. Li Y, Xu J, Chen L, Zhong WD, Zhang Z, Mi L, et al. HAb18G (CD147), a cancer-associated biomarker and its role in cancer detection. Histopathology 2009; 54: 677–687, doi: 10.1111/j.1365-2559.2009.03280.x.
https://doi.org/10.1111/j.1365-2559.2009... ). Up-regulation of CD147 also has been noted in glioma, laryngeal squamous cell, ovarian, renal cell, and skin carcinoma (³⁶36. Liang YX, He HC, Han ZD, Bi XC, Dai QS, Ye YK, et al. CD147 and VEGF expression in advanced renal cell carcinoma and their prognostic value. Cancer Invest 2009; 27: 788–793, doi: 10.1080/07357900802709167.
https://doi.org/10.1080/0735790080270916... –³⁷37. Chen X, Kanekura T, Kanzaki T. Expression of Basigin in human fetal, infantile and adult skin and in basal cell carcinoma. J Cutan Pathol 2001; 28: 184–190, doi: 10.1034/j.1600-0560.2001.028004184.x.
https://doi.org/10.1034/j.1600-0560.2001... ). CD147 was described as a marker of poor diagnosis in serous ovarian and bladder carcinomas (³⁸38. Als AB, Dyrskjot L, von der Maase H, Koed K, Mansilla F, Toldbod HE, et al. Emmprin and survivin predict response and survival following cisplatin-containing chemotherapy in patients with advanced bladder cancer. Clin Cancer Res 2007; 13: 4407–4414, doi: 10.1158/1078-0432.CCR-07-0109.
https://doi.org/10.1158/1078-0432.CCR-07... ). Collectively, CD147, the pleiotropic glycoprotein, plays a role in every stage of the progression of malignant tumors, including invasion, metastasis, angiogenesis, survival, and multidrug resistance. However, the prognostic role of CD147 in ovarian cancer is still conflicting. Given the multiple roles of CD147 in ovarian cancer development, a potential therapeutic strategy targeting CD147 to prevent ovarian cancer progression would be useful. In fact, CD147 is considered a biomarker of many tumors and is currently the subject of clinical trials in hepatocellular carcinomas (³⁹39. Chen ZN, Mi L, Xu J, Song F, Zhang Q, Zhang Z, et al. Targeting radioimmunotherapy of hepatocellular carcinoma with iodine (131I) metuximab injection: clinical phase I/II trials. Int J Radiat Oncol Biol Phys 2006; 65: 435–444, doi: 10.1016/j.ijrobp.2005.12.034.
https://doi.org/10.1016/j.ijrobp.2005.12... ). The use of CD147 as a new diagnostic and prognostic marker for bone cancer needs further study.

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