Establishment And Biological Characteristics Of Patient-Derived Xenografts For Different Grade Gliomas In NPG Mouse

Background:Human gliomas are a group of highly heterogeneous tumors with the highest incidences and poor prognosis in adult primary brain tumors.Due to the special infiltrative growth pattern and abundant neovascularization,it is difficult to completely remove the tumor by surgery,resulting in recurrence of the tumor,and in great harm to human life.In particular,the most malignant glioblastoma has a very poor prognosis with the average median survival of 14.8 months,and with the 5-year survival rate of less than 3%Gliomas originate from the glial cells of the central nervous system.Isocitrate Dehydrogenase(IDH)mutations are considered to be important early events driving glioma development.According to the IDH mutation status,1p/19q co-deletion and other genes(such as ATRX deletion,TP53 mutation),gliomas are classified into 3 major groups:IDH mutant with 1p/19q codeletion,IDH mutant with 1p/19q intact,and IDH wildtype by World Health Organization(WHO)combining the histologic and molecular features with the integrated diagnosis in 2016.Therefore,different gliomas are highly heterogeneous and complex in terms of gene mutation,malignant grading,clinical manifestations,growth patterns,sensitivity to treatment,responsiveness,and clinical outcomes,which cause to the ineffective therapies of patients with glioma.And the research and development of effective tumor-specific treatments is urgent to be required for solving this dilemmaAnimal models are the basis for studying tumor biology and treatment Traditional cancer cell line xenografts have been used both in situ and subcutaneously for several decades.However,the studies using those cell lines are being questioned because the biological characteristics of those cells are significantly different from human gliomas and cannot represent human gliomas after long-term in vitro culture.Patient-derived xenografts(PDX)are the reliable models to the biological characteristics of human gliomas.The patient-derived orthotopic xenografts(PDOX)models using patient glioma tissue digestion and short-term culture to obtain tumor cells or stem cell-like tumor cells have a high similarity of histopathologic and molecular features with the primary tumor.However,the transplanted cells from the patient tumor short-term cultures lack of the microenvironment for tumor growth,such as stromal cells such as immune cells,fibroblasts or vascular endothelial cells.The recent study of the PDOX model by fresh tissue fragments were reported a failure in implantation of intracranial gliomas.Currently,the subcutaneous PDX model for gliomas mostly focus on the GBM.The subcutaneous PDXs for combining different grade gliomas have not been studied and whether the recurrence and infiltration of xenografts have never been observedBased on the background,our research focus on the following two parts:1.After the patient's informed consent,specimens and clinical data from WHO ?-? glioma patients were collected,and fragment of gliomas were subcutaneously implanted in NOD-Prkdcscia 112rgnull(NPG)mice for glioma PDXs.And the survival of the transplanted tumor was observed by gross observation.The correlation between the tumor formation and the clinical parameters of the patients was statistically analyzed.The size of the transplanted tumor was weekly measured using vernier calipers.when tumor size reached>1.5 cm,tumors were harvested and put in transportation media,for either direct propagation into a further generation or for storage.Evaluation of the size and invasive growth characteristics of the transplanted tumor of gliomas also were carried out in our study.2 Comparison of the histological and genetic characteristics of xenografts with their parental gliomas were performed to evaluate the resemble of PDXs to their parental glioma in phenotype and genotypePart ?:The establishment of patient-derived xenografts for different grade gliomas in NPG mouseMethodSurgical specimens and clinical data of WHO ?-? glioma patients from the First Affiliated Hospital of Chongqing Medical University and obtained informed consent.The gliomas were cut into 3-5 mm size and transplanted subcutaneously into NPG mice.The survival,growth rate,and growth pattern of the transplanted tumor were analyzed,and the correlation between the survival of the transplanted tumor and the clinical parameters including gender,age,and glioma grades,size,IDH mutation status,and nuclear proliferating antigen Ki67 of glioma patients were analyzed.When the xenografts were more than 1.5 cm or two tumors were in contact with each other,the tumors were harvested and put in transportation media,for either direct propagation into a further generation or for storage.The size of xenografts tumor was measured weekly,and the tumor growth curve was drawn to evaluate the growth latency and growth characteristics of different generations of transplanted tumors.Furthermore,the recurrence and infiltrative growth were also be investigated in the current studyResultOf the 28 surgical glioma specimens,16 were effectively transplanted.Among the 16 cases of effective transplantation,there were 3 cases of WHO class ? glioma,5 cases of grade ? and 8 cases of grade ?.Statistical analysis of the correlation between clinical parameters of glioma patients and the success of transplanted tumors revealed that the formation of glioma xenografts was associated with more than 5%Ki67 expression(P=0.004)and IDH wildtype(P=0.002).The overall transplant success rate was 68.75%(11/16),of which the grade ? transplant success rate was 33.3%(1/3),the grade ? transplant success rate was 60%(3/5),and the grade ? transplant success rate was 87.5%.(7/8).Analysis of the growth curve of transplanted tumors revealed that the primary xenografts had a latent period,in which the P1 generation of WHO ? latency was 11 weeks,WHO ? latency varied between 14 and 22 weeks,and the ? ranged from 5 to 22 weeks.Interestingly,the grade ? anaplastic oligodendroglioma,IDHMUT presented two new spread xenografts near the two primarily implanted gliomas.Moreover,grade ? diffuse astrocytoma,IDHWT and grade ? glioblastoma,IDHWT presented recurrence after the removal of the tumor mass at second-generation(P2)third-generation(P3)ConclusionThe PDX model for WHO ?-? gliomas in NPG mice was established by tissue fragments transplantation.The success rate of xenografts was associated with high Ki67 expression and IDH wildtype.The success rate varied from 33.3-87.5%.After 5 to 22 weeks of latency,the rapid growth of xenografts was observed.The second generation and subsequent generations have a shorter latency or no latency.In addition,the xenografts retain the invasive and recurrent growth characteristics of their corresponding parental glioma.Part ?:Biological characteristics of glioma PDX modelMethodTo identify the biological features of xenografts,the following two aspects were investigated:(1)The histopathological and immunological feature of xenografts in serial passages were evaluated by H&E staining and by immunohistochemical or immunofluorescence staining for glioma-specific markers such glial fibrillary acidic protein,GFAP),oligodendrocyte lineage transcription factor 2(Olig-2)and vimentin(Vimentin).The anti-human and anti-mouse CD31 monoclonal antibody was used to evaluate whether there is a replacement of human microvascular by murine microvascular.The nuclear proliferation marker Ki67 was stained to analyze the proliferation ability of transplanted tumor and patient-derived gliomas by IHC.(2)Extracting the DNA from the serial xenografts and their corresponding parental glioma,IDH mutation and the stability of satellite polymorphism were investigated by Q-PCR and STR methods.ResultH&E staining results showed that WHO ?-? patient-derived xenografts maintained the heterogeneity and diversity of histological morphology in their respective grades of gliomas.But in terms of cell density,atypia,and vascular endothelial cell proliferation of xenografts are more potent than patients.Immunostaining of GFAP,Vimentin and Olig-2 showed that the transplanted tumor maintained the key immunophenotype of parental glioma.The expression of CD31 showed that CD31 expression was increased in PDX tumor tissues compared with corresponding parental gliomas,while CD31 expression was decreased in human vascular endothelial cells.Ki67 results showed that the proliferation of PDX tumor cells was significantly stronger than that of patients with glioma.IDH gene mutation detection showed that most of the PDX tumors maintained the glioma IDH gene status in patients,and except for one case of IDH1 wild-type glioblastoma harboring a R132H mutation in the P1 generation,but a recover occurred in the subsequent passage.The STR results showed that most of the transplanted tumors of each generation strictly preserved the microsatellite DNA characteristics of the patient-derived glioma,except for two cases showing the loss of gene fragments.ConclusionIn the present study,the established xenografts for WHO ?-?gliomas in NPG mice are highly consistent with their corresponding parental tumors in terms of growth pattern,histopathological and genetic characteristics,providing a reliable model for new agents screening and for patient-tailored therapy.