TTfields Inhibits Non-small Cell Lung Cancer Brain Metastases In Cerebral Organoids

Background:Non-small cell lung cancer(NSCLC)is a highly aggressive tumour type,with brain metastases as the major complication,which contribute to the poor prognosis of patients.Currently,an effective treatment for NSCLC brain metastases is lacking,and palliative treatment is often used.Tumourtreating fields(TTFields)are low-intensity,medium-frequency alternating electric fields that selectively inhibit tumour cell proliferation.Studies have shown that TTFields can reduce the proliferation rate of NSCLC cells.However,only a few studies have focused on NSCLC brain metastases.Preclinical studies mostly use cell models,which lack the support of the brain microenvironment,causing biases in the results.The efficacy,optimal frequency,and specific mechanism of TTField intervention in NSCLC brain metastases,as well as its potential negative effects on brain development,deserve research attention.Objective:This study intends to establish an in vitro NSCLC brain metastasis tumour model to clarify the effect of TTFields on NSCLC brain metastasis cells,elucidate the specific mechanism of TTField intervention in NSCLC brain metastases,and determine whether TTField intervention affects neurodevelopment of cerebral organoids(COs).Methods:1.NSCLC brain metastasis cerebral organoids(BMCOs)were constructed by implanting the tumour cells derived from patients with NSCLC brain metastases into COs.The model quality was assessed using immunofluorescence and single-cell sequencing analyses.2.Different frequencies of TTFields were applied to NSCLC brain metastasis cells and BMCO;cell viability was determined using the CCK8assay;the growth range of NSCLC cells in BMCO was observed using a fluorescence microscope;and changes in NSCLC cell clusters were analysed through single-cell sequencing.3.The effect of TTFields on the cell submicroscopic structure and organelles was observed through electron microscopy;the genes and functional molecular pathways affected by TTFields were analysed through single-cell transcriptome sequencing;JC-1 The cell membrane potential was determined experimentally,and the cellular reactive oxygen species level was determined through flow cytometry.4.After applying the TTField intervention to the normal CO,the expression levels of neural cells and neural differentiation markers were determined through immunofluorescence assay;and the changes in nervous system cell lineages were analysed through single-cell sequencing.Differential gene enrichment analysis was performed on CO and controls in the TTField-treated group.Results:1.The red fluorescence signal of tumour cells in the BMCO increased with the extension of culture time,as observed under the fluorescence microscope.The pathological sections and original tumour tissues of the BMCO organoid model were all CK(+),TTF-1(+),EGFR(+),and Ki67(+).In total,12 distinct cell types,including a cluster of EPCAM+ cells having characteristics of lung cancer brain metastasis cells,were identified using the BMCO single-cell transcriptome analysis.2.Compared with the control group,each frequency of TTFields exerted an inhibitory effect on the NSCLC tumour cells;the inhibition rates of A549 and BM104 cells at 150 k Hz reached 46.1% ± 3.1%(p < 0.001)and 31.9% ± 6.0%(p < 0.001),respectively.In BMCO,the fluorescence signal of tumour cells was weakened;the red fluorescence intensity was weakened and the area was reduced(p < 0.001).Single-cell transcriptome analysis indicated that the expression of tumour cell markers(EPCAM)in the BMCO of the TTField intervention group was significantly decreased compared with that in the control group(p < 0.001).3.The tumour cells in the TTField intervention group exhibited a larger area of necrosis in the invasion area,a significantly collapsed tissue structure,and a significantly increased level of pyknosis compared with those in the control group(p < 0.001).Transmission electron microscopy showed an increase in abnormal mitotic events,changes in mitochondrial morphology,and disappearance of mitochondrial cristae in the TTField intervention group.Scanning electron microscopy indicated that after treating BMCOs with TTFields,the tumour cells were disordered and apoptotic.The KEGG pathway enrichment analysis showed that fatty acid metabolism,cytochrome P450,glutathione metabolism,tricarboxylic acid cycle,and other pathways highly related to the mitochondrial function were significantly downregulated after TTField intervention.GO enrichment analysis indicated that TTField intervention significantly affected the expression of genes involved in mitochondrial respiratory chain,cellular respiration,and electron transport chain,and other related pathways.Flow cytometry indicated that the ROS level in the A549 and BT1009 cells increased and mitochondrial membrane potential decreased after TTField intervention.4.Multiple immunofluorescence staining showed that the expression level of the radial glial cell marker PAX6,the intermediate group neuron marker Tbr2,the hippocampal marker Frizzled9,and the midbrain marker Otx2 was significantly higher in the TTField intervention group than in the control group.The expression was not affected,and the LIM homeobox family gene Islet1 and the forebrain development-related gene EMX1 exhibited no significant changes in their expression.After TTField intervention,no significant differences were found in the expression of marker genes of neuronal cell subsets in the BMCO single-cell profile in the majority of neuronal cells.KEGG pathway enrichment analysis showed that TTField treatment did not alter the mitochondrial respiration,cell cycle,cell connectivity,and other important activities in neural cells.Cerebral cortex development and neuronal differentiation in the central nervous system were also not significantly affected,as observed using the GO analysis.Conclusion:The NSCLC BMCO metastasis organoid model constructed in vitro indicated that TTField intervention can inhibit the proliferation and invasiveness of NSCLC brain metastasis cells,and the optimal treatment frequency is 150 k Hz.Additionally,the findings suggested that TTField intervention can lead to mitochondrial dysfunction,without affecting the neurodevelopment of brain organoids.