| Brain metastases are the most common metastases in advanced non-small cell lung cancer(NSCLC),and patients with brain metastases have poor prognosis.Traditional treatments for brain metastases are ineffective.The application of immune checkpoint inhibitors(ICIs)has revolutionized the clinical practice of NSCLC treatment,and previous clinical research data suggest that patients with brain metastases from NSCLC can also benefit from the treatment of ICIs.However,the vast majority of clinical studies excluded patients with active brain metastases or who had received corticosteroids prior to immunotherapy,and the efficacy of immunotherapy in realworld NSCLC patients with brain metastases remains to be determined.Brain metastases involving different lobes of the brain lead to different neurological abnormalities,which can be life-threatening in severe cases.Therefore,it is of great clinical significance to explore the influence of brain metastases in different functional regions of the brain on the efficacy of ICIs.Studies have shown that exosomes play an important role in the process of metastases,and they can form a "pre-metastatic niche" by remodeling the microenvironment of distant target organs,which is conducive to the colonization and metastases of tumor cells to target organs.Astrocytes,as important immune and support cells in the brain,play an important role in maintaining the homeostasis of the brain microenvironment.Therefore,exploring the effect of lung cancer cell-derived exosomes on astrocytes is extremely important for in-depth understanding of the mechanism of NSCLC brain metastases.In this study,we retrospectively analyzed the incidence of brain metastases in lung adenocarcinoma patients in the real world,and explored the differences in the efficacy of immunotherapy for different brain metastases.Then lung cancer cell exosomes were co-cultured with astrocytes,cell viability was detected by cck8,and astrocyte proliferation and apoptosis were observed by Edu/Hoechst33342 and flow cytometry,and to identify key mi RNAs in exosomes of lung cancer cells,biochips were used to screen out the key elements by mi RNA sequencing technology.Combined with clinical research and basic research,the study focuses on the effect of immunotherapy on different lobes metastases in patients with brain metastases and how exosomes from lung cancer cells influence astrocytes to remodel the metastatic microenvironment in the brain.Part 1 Evaluation of the efficacy of immunotherapy in real-world lung adenocarcinoma patients with brain metastasesObjectives: To evaluate the efficacy of ICIs in patients with lung adenocarcinoma brain metastases in the real world,to analyze the incidence of brain metastases in various functional regions of the brain in lung adenocarcinoma patients and the efficacy of immunotherapy,and to detect the levels of exosomal mi RNAs in the plasma of patients with lung adenocarcinoma brain metastases.Methods: The clinical data of lung adenocarcinoma patients with brain metastases from July 2015 to November 2020 in the Department of Oncology were collected,the efficacy of immunotherapy was analyzed,and the incidence of brain metastases in each functional lobe of the brain and the immunotherapy for brain metastases in each functional lobe were explored.Cox regression model analysis included ECOG score,the presence or absence of neurological symptoms,the presence or absence of radiotherapy,and the effects of immunotherapy regimens on the effects of immunotherapy for intracranial metastases in various functional lobes.At the same time,the plasma of patients with brain metastases was collected,then the exosomes in the plasma were extracted and mi RNA sequencing was performed,and bioinformatics tools were used to analyze the expression differences of exosomal mi RNAs in such patients.Results: A total of 447 patients with NSCLC were enrolled in this study,including 198 patients with lung adenocarcinoma,including 157 patients without brain metastases and 41 patients with brain metastases.78%(32 Cases)of lung adenocarcinoma patients with brain metastases and 79.6%(125 Cases)of patients without brain metastases progressed after immunotherapy,with a median follow-up of 31.7 months.ORR was not statistically different between patients with and without brain metastases,but it was comparable(29.3% vs 43.9%;p = 0.089).Patients in the brain metastases group had lower DCR(58.5% vs 78.3%;p = 0.01)and shorter PFS(3.6 months vs 8.6 months;p = 0.069;HR=1.428;95% CI,0.924-2.207).The investigators evaluated the efficacy of intracranial lesions in 31 patients with brain metastases,of which the intracranial ORR was 35.5%,the DCR was 71.0%,and 77.4% of the patients received immunotherapy for both pulmonary lesions and intracranial lesions.Multivariate Cox regression analysis showed that patients with and without neurological symptoms had similar progression-free survival(3.0 months vs 4.4 months;p=0.553).There was no significant difference in PFS between patients who received and did not receive intracranial radiation therapy.Compared with patients receiving ICIs monotherapy,patients receiving combination therapy with ICIs had longer PFS(6.3 months vs 2.9 months;p=0.069).Further efficacy analysis of patients with brain metastases who received later-line immunotherapy showed no significant difference between the combination and monotherapy groups.Similar results were observed in patients without brain metastases.The analysis results of the incidence of brain metastases in each brain region of the patients and the efficacy of immunotherapy showed that the median PFS of patients with or without frontal lobe metastases was not statistically different(3.3 months vs 10.4 months);the median of patients with or without parietal lobe metastases has no statistical difference in PFS(3.1 months vs 5.0 months);There is no significant difference in median PFS between patients with and without temporal lobe metastases(3.4 vs 4.4 months)and no statistical difference of median PFS in patients with and without occipital lobe metastases(3.4 months vs 3.6 months).Compared with lung adenocarcinoma patients without cerebellar metastases,the median PFS of patients with cerebellar metastases was significantly shorter,which is only 2.8 months,and the median PFS of patients without cerebellar metastases was 13.8 months.Patients with extensive brain metastases who received immunotherapy had a median PFS of only 2.8 months,and patients without extensive brain metastases had a median PFS of 5.6 months after immunotherapy.They were statistically different(p=0.02).The ic PFS results of intracranial lesions in lung adenocarcinoma patients with brain metastases in response to immunotherapy showed that the median ic PFS of patients with frontal lobe metastases was about 6.5 months,while the median ic PFS of patients without frontal lobe metastases was not reached,and there was no statistical difference between the two groups groups.The median ic PFS of patients with parietal metastases was about 14.5 months,while the median ic PFS of patients without parietal metastases was not reached,and there was no statistical difference between the two groups.The median ic PFS of patients with temporal lobe metastases was about 14.5 months,while the median ic PFS of patients without parietal lobe metastases was not reached,and there was no statistical difference between the two groups.The median ic PFS of patients with occipital lobe metastases was not reached,while the median ic PFS of patients without occipital lobe metastases was about 14.5 months,and there was no statistical difference between the two groups.However,compared with lung adenocarcinoma patients without cerebellar metastases,the ic PFS of patients with cerebellar metastases was significantly shorter which was only 3.6 months,and the ic PFS was not reached in patients without cerebellar metastases.The median PFS of patients with extensive brain metastases after immunotherapy was only 6.47 months,and the ic PFS of patients without extensive brain metastases was not reached.There was no statistical difference between the two groups,but a statistical marginal effect was reached(p=0.073).Among the 15 patients with cerebellar metastases,7 patients had extensive metastases in the whole brain at the same time.After excluding 7 patients with extensive metastases in the whole brain,the median intracranial PFS in the cerebellar metastases group was 3.5 months,and the median intracranial PFS in the nocerebellar metastases group was not reached,and the difference was statistically significant(p=0.008).At the same time,we also compared the PFS of patients with lung adenocarcinoma brain metastases with or without symptoms,whether they received radiotherapy,as well as monotherapy and combination therapy groups.The median PFS of patients received immunotherapy in the radiotherapy group was 4.4 months and 3.1 months in the nonradiotherapy group,and there was no significant difference between the two groups.The median PFS was 6.3 months in the first-line combination therapy group and 2.9 months in the monotherapy group(p=0.069).The median PFS was 6.3 months and 3.0 months in the later-line combination therapy group and the monotherapy group respectively,but there was no statistical difference between the two groups.For intracranial lesions,the intracranial PFS of lung adenocarcinoma patients with brain metastases was 12.9 months.Compared with asymptomatic lung adenocarcinoma patients with brain metastases,the intracranial PFS of symptomatic patients was shorter which was only 5.6 months,but there was no statistical difference between the two groups.The median intracranial PFS was 14.5 months in the combination therapy group and 12.9 months in the monotherapy group respectively,with no statistical difference.Compared the median intracranial PFS of the radiotherapy group with the nonradiotherapy group after receiving immunotherapy,the median intracranial PFS of the radiotherapy group was 6.3 months,and the median intracranial PFS of the nonradiotherapy patients was not reached,and there was no statistical difference between the two groups.mi RNA sequencing was performed on exosomes extracted from the plasma of patients with and without cerebellar metastases,and it was found that the differentially expressed mi RNAs in the two groups were: hsa-mi RNA-331-5p,hsa-mi RNA-542-5p,hsa-mi RNA-26a-2-3p,hsa-mi RNA-99a-3p,hsa-mi RNA-184 and hsa-mi RNA-3065-5p.Conclusions: Compared to patients without brain metastases,patients with brain metastases have a poorer effect of immunotherapy,and most patients have consistent responses to immunotherapy in primary lung tumors and brain metastases.The proportion of lung adenocarcinoma brain metastases is less in the cerebellum.However,the efficacy of immunotherapy in such patients is poor.Compared with patients without cerebellar metastases,there were 6 significantly up-regulated exosomal mi RNAs in the blood of patients with cerebellar metastases.Part Ⅱ: The mechanisms of exosomes promoting brain metastases of lung cancer by regulating astrocytesObjectives: To explore the effect of lung cancer cell-derived exosomes on human astrocytes,and to deeply understand the mechanism of promoting NSCLC brain metastases by regulating astrocytes.Methods: The exosomes secreted by lung cancer cell line H1299 cell line were extracted and co-cultured with human astrocytes cell line SVG P12.Cell viability was detected by cck8,and astrocyte proliferation and apoptosis were observed by Edu/Hoechst33342 and flow cytometry.Biochip was used to screen out the key apoptotic proteins,and the mi RNA sequencing technology was used to identify the key mi RNAs in the exosomes of lung cancer cells.Results: The exosomes secreted by H1299 cell line were extracted and co-cultured with human astrocyte cell line SVG P12.The results of CCK8 showed that after the addition of exosomes from H1299 cell line,the cell viability of SVG P12 decreased,and the cell viability decreased to 50% when the exosome protein concentration was 60ug/ml.Flow cytometry results showed that H1299 cell line exosomes could promote the apoptosis of SVG P12 cell line.Edu/Hoechst33342 further confirmed the above results.After SVG P12 cell line was treated with H1299 cell line exosomes for 24 hours,the cytokines in the cell supernatant changed.Compared with the control group,GROα/CXCL1,IFN-γ,IL-3,IL-5,IL-15,LIF,M-CSF,NGF,PDGF and VEGF increased,and the level of IL-7 decreased,with significant statistical differences.Labelfree non-scalar proteomics found that the expressions of MAP2K1,TUBA1 C,RELA,and CASP6 in apoptosis-related pathways increased,and there were statistical differences.After comparing the differentially expressed mi RNAs in the exosomes of H1299 cell line and normal lung bronchial epithelial cell line Beas-2b,it was found that the differentially expressed and statistically significant hsa-mi R-140-3p may participate in apoptosis process of SVG P12 cell line which was induced by H1299 cell line exosomes by regulating the MAP2K1 signaling pathway.Conclusions: H1299 exosomes disrupt the stability of the brain microenvironment by inducing astrocyte apoptosis,which is one of the mechanisms promoting brain metastases of NSCLC. |
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