Background/Aims: Gap junctions, which are assembled by connexins, can directly connect the cytoplasm of adjacent cells and enable gap junctional intercellular communication (GJIC) as well as metabolic coupling between neighboring cells. Here, we investigated the role of connexin 43 (Cx43) and its derived GJIC in the interplay between non-small cell lung cancer (NSCLC) cells and cancer-associated fibroblasts (CAFs). Methods: CAFs and NSCLC cells were co-cultured with direct contact and separated using flow cytometry. Glucose uptake, lactate production, and the expression and activity of PKM-2 and LDH-A in sorted CAFs were measured by a colorimetric assay, western blotting, and enzyme-linked immunosorbent assay (ELISA). Meanwhile, E-cadherin and N-cadherin expression and the migration and invasion of sorted NSCLC cells were detected by western blotting, wound width, and Transwell assays. Pyruvate, acetyl-CoA, and citric acid levels, ATP levels, and LDH-B and α-KG activity in sorted NSCLC cells were determined by a colorimetric or fluorometric assay and ELISA, respectively. Functional GJIC between cells and the subcellular location of connexins were detected by a “Parachute” assay and immunofluorescence. Levels of α-SMA, Cx43, and LDH-B in tissue from patients with NSCLC were determined by immunohistochemistry. Results: Cx43 accumulated in the plasma membrane, which favored the assembly of asymmetric unidirectional GJIC from CAFs to NSCLC cells. CAFs underwent increased aerobic glycolysis and promoted the epithelial-mesenchymal transition, migration, and invasion of NSCLC cells. In contrast, NSCLC cells experienced enhanced oxidative phosphorylation upon CAF stimulation, with an increase in ATP generation and thereby activation of the PI3K/Akt and MAPK/ERK pathways. Metabolic coupling between CAFs and NSCLC cells was under the strict control of Cx43-formed unidirectional GJIC. Patients with high tri-expression of α-SMA, Cx43, and LDH-B had the shortest overall survival and relapse-free survival compared with those with individual overexpression or high bi-expression. Conclusion: Cx43-formed unidirectional GJIC plays a critical role in mediating close metabolic cooperation between CAFs and NSCLC cells to support the malignant progression of NSCLC.
BackgroundDuring human pregnancy, placental trophectoderm cells release extracellular vesicles (EVs) into maternal circulation. Trophoblasts also give rise to cell-free DNA (cfDNA) in maternal blood, and has been used for noninvasive prenatal screening for chromosomal aneuploidy. We intended to prove the existence of DNA in the EVs (evDNA) of maternal blood, and compared evDNA with plasma cfDNA in terms of genome distribution, fragment length, and the possibility of detecting genetic diseases.MethodsMaternal blood from 20 euploid pregnancies, 9 T21 pregnancies, 3 T18 pregnancies, 1 T13 pregnancy, and 2 pregnancies with FGFR3 mutations were obtained. EVs were separated from maternal plasma, and confirmed by transmission electronic microscopy (TEM), western blotting, and flow cytometry (FACS). evDNA was extracted and its fetal origin was confirmed by quantitative PCR (qPCR). Pair-end (PE) whole genome sequencing was performed to characterize evDNA, and the results were compared with that of cfDNA. The fetal risk of aneuploidy and monogenic diseases was analyzed using the evDNA sequencing data.ResultsEVs separated from maternal plasma were confirmed with morphology by TEM, and protein markers of CD9, CD63, CD81 as well as the placental specific protein placental alkaline phosphatase (PLAP) were confirmed by western blotting or flow cytometry. EvDNA could be successfully extracted for qPCR and sequencing from the plasma EVs. Sequencing data showed that evDNA span on all 23 pairs of chromosomes and mitochondria, sharing a similar distribution pattern and higher GC content comparing with cfDNA. EvDNA showed shorter fragments yet lower fetal fraction than cfDNA. EvDNA could be used to correctly determine fetal gender, trisomies, and de novo FGFR3 mutations.ConclusionsWe proved that fetal DNA could be detected in EVs separated from maternal plasma. EvDNA shared some similar features to plasma cfDNA, and could potentially be used to detect genetic diseases in fetus.
Objective: The present study aimed to evaluate the efficacy of a non-invasive prenatal test (NIPT) in the detection of the sex chromosome aneuploidies (SCAs) at our prenatal diagnosis centre.Methods: Among a cohort of 34,717 pregnancies, maternal plasma samples from our prenatal diagnosis centre were subject to analysis of SCAs using NIPT detection.Pregnant women with NIPT positive results of SCAs were recommended to undergo an invasive prenatal diagnosis (i.e. karyotyping and fluorescence in situ hybridization) to validate the prediction value of NIPT.Results: From 34,717 clinical pregnancies, 229 (0.66%) pregnancies were identified with SCAs. Of these, 78 (34.1%) cases were positive for 45,X and 151 (65.9%) cases comprised a sex chromosome trisomy. Of the 229 positive NIPT results, 193 (84.3%) cases had accepted an invasive diagnosis involving karyotyping analysis of the amniotic fluid, which confirmed 67 cases (34.7%) as true positive, as well as 126 cases (65.3%) as false positive. The positive predictive values were 23.07%, 50%, 36% and 27.27% respectively. The remaining 36 (15.7%) cases declined a prenatal diagnosis.
Cell-free DNA (cfDNA) testing for common fetal trisomies (T21, T18, T13) is highly effective. However, the usefulness of cfDNA testing in detecting other chromosomal abnormalities is unclear. We evaluated the performance of cfDNA testing for genome-wide abnormalities, and analyzed the incremental yield by reporting extra abnormalities. We performed genome-wide cfDNA testing in 15,626 consecutive pregnancies prospectively enrolled in this study. cfDNA testing results were reported and counseling was given depending on the presence of extra chromosomal abnormalities. cfDNA testing identified 190 cases (1.2%) of chromosomal abnormalities including 100 common trisomies and 90 additional abnormalities. By expanding the cfDNA reporting range to genome-wide abnormalities, the false positive rate increased to 0.39% (P<0.001) and positive predictive value (PPV) was reduced to 65.58% (P=0.42). However, the detection yield increased from 0.44% to 0.65% (P=0.014), and cfDNA testing detected 38.61% (39/101) additional abnormalities with no ultrasound and biochemical screening findings. cfDNA testing outperformed biochemical screening by showing 60 times higher true positive rate and fewer false negative results. Genome-wide cfDNA testing significantly increased the diagnostic yield by detecting extra abnormalities, especially those without diagnostic indications. Genome-wide cfDNA testing has fewer false positive and false negative results compared with biochemical screening.
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