Glycogen synthase kinase 3 inhibition lowers PD-1 expression, promotes long-term survival and memory generation in antigen-specific CAR-T cells.

Successful remission in hematological cancers by CAR-T cell immunotherapy has yet to be replicated in solid tumors like GBM. A significant impediment of CAR-T immunotherapy in solid tumors is poor exposure of T cells to tumor antigens resulting in suboptimal CAR-T cell activation, which ultimately fails to induce a robust anti-tumor immune response. Costimulatory moieties in advanced-generation CARs, along with additional IL2 therapy has been shown to be insufficient to overcome this hurdle and have its cytotoxic limitations. GSK3 is constitutively active in naïve T cells and is transiently inactivated during T cell activation resulting in rapid T cell proliferation. Pharmacologic inhibition of GSK3 in GBM-specific CAR-T cells reduced FasL expression, increased T cell proliferation and reduced exhaustion by lowering PD-1 levels resulting in the development of CAR-T effector memory phenotype. Treatment with GSK3-inhibited CAR-T cells resulted in 100% tumor elimination during the tumor-rechallenge experiment in GBM-bearing animals and increased accumulation of memory CAR-T cells in secondary lymphoid organs. These adjuvant-like effects of GSK3 inhibition on activated CAR-T cells may be a valuable adjunct to a successful implementation of CAR-T immunotherapy against GBM and other solid tumors.

Inertial focusing for tumor antigen-dependent and -independent sorting of rare circulating tumor cells.

Circulating tumor cells (CTCs) are shed into the bloodstream from primary and metastatic tumor deposits. Their isolation and analysis hold great promise for the early detection of invasive cancer and the management of advanced disease, but technological hurdles have limited their broad clinical utility. We describe an inertial focusing-enhanced microfluidic CTC capture platform, termed "CTC-iChip," that is capable of sorting rare CTCs from whole blood at 10(7) cells/s. Most importantly, the iChip is capable of isolating CTCs using strategies that are either dependent or independent of tumor membrane epitopes, and thus applicable to virtually all cancers. We specifically demonstrate the use of the iChip in an expanded set of both epithelial and nonepithelial cancers including lung, prostate, pancreas, breast, and melanoma. The sorting of CTCs as unfixed cells in solution allows for the application of high-quality clinically standardized morphological and immunohistochemical analyses, as well as RNA-based single-cell molecular characterization. The combination of an unbiased, broadly applicable, high-throughput, and automatable rare cell sorting technology with generally accepted molecular assays and cytology standards will enable the integration of CTC-based diagnostics into the clinical management of cancer.

Effect of impeller speed and pH on the production of poly(3-hydroxybutyrate) using Bacillus cereus SPV.

P(3HB), is one of the most well studied polyhydroxyalkanoates. It is biodegradable, biocompatible, exhibits thermoplastic properties and can be produced from renewable carbon sources. The commercial exploitation of P(3HB) has been mainly held back by its high production costs. Hence, a lot of research is required to optimize P(3HB) fermentation conditions. In this study we have focused on the effects of impeller speed and pH on P(3HB) production in Bacillus cereus SPV. Four different impeller speeds, 50, 125, 250, and 500 rpm, were used. The highest amount of P(3HB) accumulation was achieved using 125 rpm impeller speed (34% dcw) and this was attributed to optimal cell growth rate. Also, pH-stat fermentations were carried out at pH 3.0, 6.8, and 10. This study confirmed that lack of P(3HB) degradation during unbuffered Bacillus fermentations is due to the low pH conditions. This observation is crucial for the industrial exploitation of the genus Bacillus for the production of P(3HB).