Relationship Between TrKB Overexpression And Anoikis Resistance Of Osteosarcoma Cells And Its Effect On Growth And Metastasis Of Osteosarcoma

Osteosarcoma is one of the most common primary malignant tumors of bone in adolescents. High incidence rate of lung metastasis mainly induces the poor prognosis of this malignant tumor. 80% of osteosarcoma patients have lung metastasis when they are diagnosed as osteosarcoma. In recent 25 years, there has been great development in the therapy of osteosarcoma. Near 80% of osteosarcoma patients are able to retain diseased limbs and 5-year survival outcomes are increased from about 20% to 80%. However, more than half of osteosarcoma patients still die from metastasis or relapse.Metastasis includes a multitude of complex events, such as tumor cell invasion to the surrounding tissue, intravasation to lymphatics or the vasculature, transporting through, and extravasation from, these vessels, seeding at a distant site and forming metastasis. In order to successfully metastasize, tumor cells must sustain a number of genetic or epigenetic alterations that allow them to complete the various steps in the process. For example, metastasizing tumor cells must acquire the ability of anoikis resistance when detach from the adhesion to other cells or the extra cellular matrix (ECM). Resistance to anoikis has been suggested to be the first prerequisite for cancer cells to metastasize.Anoikis is a special type of apoptosis. It occurs when normal epithelial cells or non-metastatic solid tumors detach from home position into blood flow. The significance of anoikis lies in preventing the implanting and growing of detached cell at improper locations. However, tumor cells, especially some metastatic malignant tumor cells, are capable of anoikis resistance, thus escape apoptosis when detach from the primary tumor and succeed in metastasis. Anoikis resistance has been improved in tumors generated from epithelial tissue such as lung cancer, intestinal cancer, malignant melanoma and so on. Anoikis resistance occurs not only in cancers but also in sarcomas. Our previous studies and some others indicated anoikis resistance also happened in osteosarcoma.TrKB is a neurotrophic receptor tyrosine kinase which plays an important role in the development and function of neural system, binding with its ligand BDNF. However, more and more studies indicate that overexpression of TrKB is closely associated with tumorigenesis and metastasis in a variety of human malignant tumors, including hepatic carcinoma, lung cancer, thyroid cancer, prostate cancer, neuroblastoma and so on. However, whether TrKB is associated with anoikis resistance of osteosarcoma or not and how it works? There still have been short of associated studies.Our study contains four successive parts. The first part was to evaluate the expression of TrKB in osteosarcoma tissues and different osteosarcoma cell lines by immunohistochemical and immunofluorescence assay. The second part was to acquire anoikis-resistant osteosarcoma cell model and subsequently evaluating various key cellular steps leading to tumorigenesis and metastasis using cell-based assays. The third part was to evaluate TrKB mRNA and protein levels in osteosarcoma anoikis-resistant cells and different osteosarcoma cell lines and their tumorigenic abilities, then examine TrKB expression in tumor tissue of nude mice. The last part is to preliminarily investigate the role of TrKB in invasion and metastasis of osteosarcoma.The main results are as follows:1. By immunohistochemical and immunofluorescence assays: Expression of TrKB was detected in a greater proportion of osteosarcoma (92.0%, 46/50) than that of osteochondroma (5.9%, 1/17), P < 0. 05. The expression levels of TrKB were not related to age, gender, and pathological-type in osteosarcoma (P > 0. 05) but significantly increased in infiltration edges and relapse cases. TrKB expression showed significantly higher in MTF cell line (high tumorigenic and high metastatic) than in SaOS-2 cell line (non-tumorigenic).2. Anoikis rates increase in a time-dependent manner in a time horizon. Anoikis rate in MTF cells began relatively rapidly after prevention of adhesion and decreased quickly. After cell suspended for 72h, anoikis rate of MTF cells achieved stable state. On the other hand, anoikis rate of suspended SaOS-2 cells increased in a time-dependent manner. In a word, MTF cells are higher of anoikis resistance than SaOS-2 cells. We succeeded in establishing MTF anoikis-resistant cell model by special cultural method but failed in establishing cell model using SaOS-2 cell line. The re-adherent MTF cell was considered as anoikis-resistant cell model.3. An obvious enhancement of cell proliferation in MTF anoikis-resistant cells was observed by MTT assay but the change between adherent and re-adhenent SaOS-2 cells was not significant.4. MTF anoikis-resistant cell mobility (62±15.9) is obviously higher than adherent (3.6±6.3), but there is no difference between adherent and re-adherent SaOS-2 cells.5. The level of TrKB mRNA and protein expression appears to be much higher in MTF anoikis-resistant cells than in adherent MTF cells but it is almost no difference between adherent SaOS-2 and re-adherent or even lower in the latter.6. MTF cells are of high malignancy and tumorigenic capacity. MTF anoikis-resistant cells were even capable of subcutaneous metastasis. SaOS-2 is a type of low potential malignant and nontumorigenic cell line. Both adherent SaOS-2 cells and re-adherent SaOS-2 cells were nontumorigenic.7. It is positive in both MTF cells injected subcutaneous tumor and MTF anoikis-resistant cells injected subcutaneous primary and metastatic tumor tissues, by TrKB immunostaining. However, TrKB expression in MTF anoikis-resistant cells injected subcutaneous tumor was significantly higher than in MTF cells injected subcutaneous tumor, and TrKB expression in MTF anoikis-resistant cells injected subcutaneous metastatic tumor tissue was the highest of all.8. MTF cells were treated by specific tyrosine kinase inhibitor K252a for 24h. Treated MTF cells turned round, aggregated, even fused. However, negative control MTF cells were polygon, scattered, sharpness of border, and full of link to each other.9. Compared to untreated group, MTF cells'invasive abilities were significantly weakened (2.29±1.11/76.29±14.61, P < 0. 05).10. Compared to untreated group, MTF cells'microfilaments shortened, retracted and arranged disorderly.The main conclusions are as follows:1. TrKB overexpression can be regarded as an independent risk factor in the evaluation of osteosarcoma's biological behaviors and may play an important role in tumorigenesis, growth and metastasis of osteosarcoma.2. The ability of anoikis resistance is variable in different osteosarcoma cell lines and our method of establishing anoikis-resistant osteosarcoma cell model is only available for the osteosarcoma cell line which is high of anoikis resistance.3. There is an obvious enhancement of proliferation, migration and tumorigenic ability in anoikis-resistant osteosarcoma cells.4. Osteosarcoma cell line with high expression of TrKB is high of anoikis-resistant ability and overexpression of TrKB may enhance growth and metastasis of osteosarcoma by inhibiting anoikis.5. TrKB may function in invasion and metastasis of osteosarcoma by regulating cytoskeleton. Blockage of TrKB in MTF cell by specific tyrosine kinase inhibitor K252a may change the microfilaments structure thereby remodel the shape and weaken the in vitro invasive ability of MTF cells.