| Object:Non-small cell lung cancer (NSCLC) is a serious disease with poor prognosis. Recently, the target-direct treatment has become an important stratagem in NSCLC therapy. Epidermal growth factor receptor,(EGFR) belongs to tyrosine kinase receptor and plays a critical role in proliferation, migration and differentiation of cancer cells. EGFR is highly expressed in many cancers including lung cancer, breast cancer and so on, which is related to its gene mutation. EGFR mutation also exists in a group of NSCLC patients. Gefitinib on behalf of EGFR-TK inhibitors has a great therapeutical effect on these patients with EGFR mutation. Research revealed that mutation site always exists at exon19and exon21. But most researches only focus on single point mutation, using the cell model in vitro, which can not repeat the environment in vivo. In the present study, for the first time, we created the NSCLC mouse model harboring EGFR L858R+V834L mutation. After that, this model was used in therapeutical effect research of gefitinib.Method:Primary tumor sample was got from an in-hospital NSCLC patient, a54years old female, with the mild-middle differentiated lung cancer. Gene sequencing confirmed the genotype of the patient was EGFR L858R+V834L double mutation. Primary tumor tissue was cut to fragment sized1.0～1.5mm3. Four sites on the back of female SCID mice were chosen and two pieces fragments were transplanted to each site, which was set as the first generation (P1).When the diameter of transplanted tumor was longer than1cm, the mouse was disposed and the tumor was separated for next generation transplant, which was set as P2. Following the same method, P3and P4mice were built. Tumors from all four generations’mice were separated for paraffins section assay. EGFR expression was detected by immunohistochemistry and immunoblotting. Gene sequencing was operated in the products, which were got from DNA samples from tumor tissues through PCR. Furthermore, when the diameter of tumor was longer than4-5mm, five P3mice were given gefitinib for two weeks treatment. The size of tumor was measured every three days for evaluation of therapeutical effect.Results:1. Transplanted tumor model was built successfully. Successful rate of every generation was50%、50%、100%and87.5%for P1, P2, P3and P4respectively.2. EGFR L858R protein expressed positively in every generation.3. Tumor tissues from every generation harbored EGFR L858R+V834L double mutation at exon21site, which were as same as the DNA sequence in primary tumor tissue. 4. Gefitinib significantly inhibited tumor growth in the mouse model harboring EGFR L858R+V834L double mutation.Conclution:In the present study, we successfully built the xenograft mouse model of NSCLC with EGFR L858R+V834L double mutation for the first time. The model is sensitive to gefitinib treatment and can be used for drug screening which is targeted on EGFR mutation in NSCLC patients. Objects:Target directed treatment has become an important stratagem in NSCLC therapy, for example gefitinib on behalf of EGFR-TKI has a great clinical outcome in NSCLC patients. But there are still some patients having low sensitivity to EGFR-TKI and the drug resistant occurs gradually, which prompt people to search new therapeutical targets. In recent years, Anaplastic lymphoma kinase(ALK) fusion gene, especially EML4-ALK fusion gene has become a hot spot in NSCLC target-directed treatment research. Crizotinib on behalf of ALK inhibitor has an effective effect on NSCLC patient with ALK fusion gene, but drug resistance always occurs Whiting one year after crizotinib treatment. The underlying mechanism remains unknown. Although a study reported that in6patients, new second grade mutation in kinase domain of ALK or expand of ALK fusion gene was found, it is still unknown whether it is the reason for drug resistance. Besides, muti-resistance has been found in patients, which makes it much more complex underlying the drug resistance. In the present study, we aim to build the NSCLC mouse model harboring EML4-ALK fusion gene for the first time. Based on the model, we plan to further induce crizotinib resistance model for exploring new approaches of drug development in NSCLC patients with acquired crizotinib Resistance.Method:Primary tumor sample was got from an in-hospital NSCLC patient, a40years old male, with poorly differentiated glandular cancer. Gene sequencing confirmed the genotype of the patient was EML4-ALK fusion gene. Primary tumor sample was cut to1.0～1.5mm3sized fragment. Four sites on the back of female SCID mice were chosen and two pieces of fragments were transplanted to each site, which was set as the first generation (P1). When the diameter of transplanted tumor was longer than1cm, mouse was disposed and the tumor was separated for next generation transplant, which was set as P2. Following the same method, P3mouse were built. Tumor from all four generation mice were separated for paraffins section assay. ALK expression was detected by immunohistochemistry and immunoblotting. Gene sequencing was runned out in RNA samples from tumor tissues. When the diameter of tumor was longer than9-11mm, six P3mice were given crizotinib for two weeks treatment. The size of tumor was measured every three days for evaluation of therapeutical effect. Furthermore, crizotinib was given for as long as six month to induce drug-resistance, proteins were harvested from the tumor with crizotinib-resistance for immunoblotting assay.Results:1. The mouse model of NSCLC with EML4-ALK fusion gene was built successfully. The success rate was50%、100%and86.7%in P1, P2and P3mice respectively. 2. ALK protein expressed positively in every generation.3. Tumor tissues from every generation harbored EML4-ALK fusion gene, which was as same as the DNA sequence in primary tumor tissue.4. Crizotinib significantly inhibited tumor growth in the mouse model harboring EML4-ALK fusion gene.5. Phosphorylation level of Akt and ERK was significantly increased in tumor with acquired crizotinib resistance.Conclution:In the present study, we successfully built the transplanted NSCLC mouse model with EML4-ALK fusion gene for the first time. The model is sensitive to crizotinib and acquires resistance can be induced in the model, which can be used for further drug resistance research. |
PDF Full Text Request