Demethylating Treatment In Combination With Adoptive T-Cell Therapy Against Osteosarcoma

Osteosarcoma is the most common primary malignancy originated from bone,which usually occurs in children and adolescents and features with propensity for local invasion and early metastases.Although the five-year survival of patients receiving therapeutic regimens involving neo-adjuvant chemotherapy,surgery and post-operative chemotherapy has reached 60-80%,it will be very poor if tumor relapse or chemoresistance occurs.Besides,the core agents used in first-line chemotherapy regimen(methotrexate,doxorubicin,and cisplatin;MAP regimen)have not been changed for decades;there is hardly any mothod for patients suffering chemoresistance.Thus,the development of novel therapies except for surgical resection,chemotherapy or radiotherapy against osteosarcoma is especially urgent.Immunotherapy is well known for its role as "the fourth therapy",which is different from three conventional strategies.Adoptive cell therapy,one of the key methods of immunotherapy,has achieved great success in the latest clinival trials against leukemias.However,highly tumor-specific and strong expressed antigens are rare in solid tumor,and the microenvironment of solid tumors protected tumor cells from immune attack.Osteosarcoma also shares these features.Therefore,safe and effective adoptive immunotherapy for osteosarcoma is of great value.Cancer/testis antigens almost exclusively express in malignant tumors and immune-privileged zones,characteristic of high specificity.However,their expressing rate in osteosarcomas differ a lot among individuals,along with weakened or even silenced expression as the result of hypermethylation.Furthermore,tumor cells can impair the expression of multiple tumor suppressor genes via abnormal hypermethylation,as well as weaken the expression of some roles which participate in immune response.In this study,we utilized demethylating agent Decitabine in the treatment of osteosarcoma cells,and discovered that the expression of cancer/testis antigens in osteosarcoma cells was dramatically elevated.Importantly,the strengthened expression of cancer/testis antigens was successfully recognized by ex-vivo incubated specific CD8+ T-cells.In addition,anti-tumor effects were monitored when performing demethylating treatment alone,which were further strenghthened when used in combination with T-cell transfer treatment.However,the single-agent anti-tumor effects of demethylating treatment using Decitabine was modest.Significant anti-tumor response was monitored only when demethylating treatment was used in combination with T-cell adoptive transfer.We also established animal models with homo osteosarcoma xenografts in immune deficient mice and evaluated demethylating treatment and adoptive immunotherapy with human CD8+ T-cells.In vivo imaging system was utilized to monitor the distribution of infused CD8+ T-cells in the mouse.We found that the transferred cancer/testis antigen-specific CD8+ T-cells could home to tumor sites only when the mice received demethylating pre-treatment.Also,the efficient anti-tumor effect of combination treatment was validated by measuring tumor growth and weighting tumor tissues.Of note,we also established syngeneic immune component mouse models to evaluate the affects of demethylating treatment on immune response in the tumor.We proved that demethylating treatment had the effects of promoting lymphocyte infiltration intro the tumor and elevating the activity of intratumoral CD8+ T-cells,which was dose-dependent.We further studies potential underlying mechanism that demetylating treatment promote lymphocyte infiltration into the tumor,and discovered that it was possiblely correlated with the expression of chemokine CXCL12.CXCL12 was hypermethylated in osteosarcoma,and consequently weakened CXCL12 expression and impaired T-cell homing.Demethylating treatment elevated the expression of CXCL12 and provided optimal environment for T-cell homing to the tumor,Taken together,our results revealed that demethylating treatment could promote immune response in osteosarcoma,and induce the recognition of cancer/testis antigen-specific CD8+ T-cells to osteosarcoma cells,and consequently control osteosarcoma progression.