Osteosarcoma Organoid Construction And Its Application In GPC3-targeting Personalized Osteosarcoma Therapy

Background:Osteosarcoma(OS)is a highly aggressive malignant bone tumor.Drug resistance and the lack of molecular therapeutic targets are pressing issues in the clinical treatment of OS.Patientderived organoids(PDOs)are three-dimensional structures derived from patient tissues.PDOs faithfully preserve the cellular and molecular characteristics of their tissue of origin,making them an ideal experimental and therapeutic model.The construction of PDOs from OS tissue remains rarely reported.Therefore,in this study,we employed next-generation sequencing(NGS)technology to identify genetic alterations associated with OS recurrence and chemotherapy resistance.Simultaneously,we established a biobank of OS PDOs to evaluate the chemosensitivity of each PDO case and its clinical relevance.Utilizing the PDOs as an ex vivo model,we analyzed the tumor biology and targeted therapeutic implications of the identified genetic mutations detected by NGS.The aim of this research is to provide novel approaches for improving the current clinical treatment of OS by creating a biobank of OS PDOs and investigating the biological and targeted therapeutic significance of the detected gene mutations using PDOs as an ex vivo model.Methods:This study collected specimens from 24 osteosarcoma patients and performed nextgeneration sequencing(NGS)and three-dimensional cultivation of patient-derived organoids(PDOs).The response of PDOs to osteosarcoma drugs was evaluated,and cell inhibition rate scores(CIRS)were calculated to assess the patients’ clinical response to neoadjuvant chemotherapy.Additionally,biopsy tissues from the primary site and metastatic lesion of one osteosarcoma patient were collected for NGS sequencing and PDO cultivation.Mutations were confirmed by sanger sequencing,and the inhibitory effects of anti-GPC3 antibodies were observed in both PDX tumor tissue and PDOs.Furthermore,immunohistochemical staining analysis using tumor tissue microarray paraffin cores was conducted to investigate the expression of GPC3 and CD 133,as well as the efficacy of anti-GPC3 immunotherapy.Ultimately,we proved that the GPC3 antibody plays an anti-tumor effect by inhibiting the Wnt/β-catenin pathway through methods such as immunofluorescence and Western Blot.Results:This study collected 32 osteosarcoma(OS)tissue samples from 24 patients(18 preneoadjuvant chemotherapy and 14 post-neoadjuvant chemotherapy),including 8 paired patient samples.The organoid formation rates for pre-neoadjuvant and post-neoadjuvant samples were 95%and 91.7%,respectively.All cultured organoids expressed osteosarcoma marker proteins:SOX9 and Vimentin.Drug sensitivity analysis based on organoids showed varying responses to first-line osteosarcoma drugs(Doxorubicin,Cisplatin,Methotrexate,and Ifosfamide)before neoadjuvant chemotherapy.However,three cases remained capable of forming organoids and showed resistance to the drugs used in neoadjuvant chemotherapy,highlighting the necessity to optimize postoperative adjuvant treatment strategies.Next-generation sequencing of OS tissues from one patient with primary and metastatic lesions revealed four gene mutations(GPC3,SOX10,MDM4,and MAPK8)in the primary tumor,while three mutations(GPC3,SOX10,and EGF)were retained in the metastatic tumor.Notably,the abundance of GPC3 mutations in the metastatic tumor was 1.54 times that of the primary tumor.Sanger sequencing of GPC3 transcript-guided confirmed a mutation at position 1046 in exon 4.Immunohistochemical staining demonstrated significantly upregulated GPC3 expression in the metastatic tumor tissue and its organoids compared to the primary tumor.The inhibition rates of metastatic osteosarcoma organoids by 2 μg/mL anti-GPC3 antibody and its combination with cisplatin were 76.43%and 92.93%,respectively.However,except for the positive control(OS08),no GPC3 mutations were found in the remaining 23 cases tested.Immunohistochemical staining of GPC3 based on OS tissue microarray showed GPC3 expression in 73.77%(45/61)of osteosarcomas,categorized as weak(+;29/45),moderate(++;8/45),and strong(+++;8/45)levels.The intratumoral distribution of GPC3-positive cells was variable in the focal(+;10%30%;8/45),partial distribution(++;31%-70%;22/45),and most positive patterns(+++;>71%;15/45),which correlated with CD133 immunolabeling(P=9.89×10-10).Immunotherapy with anti-GPC3 antibodies was effective only in GPC3-positive OS organoids,not GPC3-negative ones.IHC staining showed that compared to GPC3-negative PDO and PDX,anti-GPC3 antibodies effectively inhibited Wnt/β-catenin signaling and induced apoptosis in GPC3positive PDO and PDX cells.Conclusions:1.Osteosarcoma organoids and PDXs well maintained the pathological and molecular properties of their derived tumors.2.Prospective drug sensitivity analysis of osteosarcoma organoids showed that weighted cell inhibition rate score(CIWS)was significantly associated with clinical therapeutic efficacy.3.GPC3 is an important gene in the formation and progression of osteosarcomas.GPC3 protein is widely distributed in osteosarcoma tissues and is positively correlated with the tumor marker CD 133.4.GPC3-targeting therapy of osteosarcomas can effectively inhibit Wnt/β-catenin signaling and induce apoptosis in GPC3-positive PDO and PDX cells.5.Immunotherapy targeting GPC3 may be a new approach for individualized treatment of OS.