LRP6 Knockout Cell Lysate

BackgroundNeural tube defects (NTDs), the second most frequent cause of human congenital abnormalities, are debilitating birth defects due to failure of neural tube closure. It has been shown that noncanonical WNT/planar cell polarity (PCP) signaling is required for convergent extension (CE), the initiation step of neural tube closure (NTC). But the effect of canonical WNT//-catenin signaling during NTC is still elusive. LRP6 (low density lipoprotein receptor related proteins 6) was identified as a co-receptor for WNT/-catenin signaling, but recent studies showed that it also can mediate WNT/PCP signaling. MethodsIn this study, we screened mutations in the LRP6 gnee in 343 NTDs and 215 ethnically matched normal controls of Chinese Han population. ResultsThree rare missense mutations (c.1514A>G, p.Y505C); c.2984A>G, p.D995G; and c.4280C>A, p.P1427Q) of the LRP6 gene were identified in Chinese NTD patients. The Y505C mutation is a loss-of-function mutation on both WNT/-catenin and PCP signaling. The D995G mutation only partially lost inhibition on PCP signaling without affecting WNT/-catenin signaling. The P1427Q mutation dramatically increased WNT/-catenin signaling but only mildly loss of inhibition on PCP signaling. All three mutations failed to rescue CE defects caused by lrp6 morpholino oligos knockdown in zebrafish. Of interest, when overexpressed, D995G did not induce any defects, but Y505C and P1427Q caused more severe CE defects in zebrafish. ConclusionOur results suggested that over-active canonical WNT signaling induced by gain-of-function mutation in LRP6 could also contribute to human NTDs, and a balanced WNT/-catenin and PCP signaling is probably required for proper neural tube development. Birth Defects Research 110:63-71, 2018. (c) 2017 Wiley Periodicals, Inc.

Background Cellular immunotherapies, such as chimeric antigen receptor modified-T cell (CAR-T) therapy, offers excellent potential for tumor treatment. The memory phenotype of CAR-T has been correlated positively with a therapeutic effect on and prognosis of cancer. Method The proliferation rates of novel CAR-T was determined by cell counting. The phenotypes of CAR-T cells were then detected by flow cytometry. The cell cytotoxicity against tumor cellsin vitrowas investigated by lactate dehydrogenase assay and luciferase assay. The cytokines secreted during these assays were determined by the cytometric bead array assay. The antitumor abilityin vivowas evaluated in NOG mice. Results Co-expression of an LRP6 full-length protein with anti-CD19 CAR significantly improved the memory phenotype of CAR-positive T-cells by enhancing the wnt signaling pathway. As compared with anti-CD19 CAR-T, anti-CD19 CAR-T-LRP6 exhibited more robust cytotoxicity against tumor cellsin vitroandin vivo, albeit fewer cytokines were releasedin vitro. Moreover, the longer survival rate and robust expansionin vivoof anti-CD19 CAR-T-LRP6 cells were found to be effective in inhibiting cancer recurrence. Conclusions CAR co-expressed with LRP6 could sustain the memory phenotype that enabled permanent relief and may further assist in the development of potent and durable T-cell therapeutics.