Breast cancer is the most frequently diagnosed malignant neoplasm and the second leading cause of cancer death among women. Epithelial-to-mesenchymal Transition (EMT) plays a critical role in the organism development, providing cell migration and tissue formation. However, its erroneous activation in malignancies can serve as the basis for the dissemination of cancer cells and metastasis. The Zeb1 transcription factor, which regulates the EMT activation, has been shown to play an essential role in malignant transformation. This factor is involved in many signaling pathways that influence a wide range of cellular functions via interacting with many proteins that affect its transcriptional functions. Importantly, the interactome of Zeb1 depends on the cellular context. Here, using the inducible expression of Zeb1 in epithelial breast cancer cells, we identified a substantial list of novel potential Zeb1 interaction partners, including proteins involved in the formation of malignant neoplasms, such as ATP-dependent RNA helicase DDX17and a component of the NURD repressor complex, CTBP2. We confirmed the presence of the selected interactors by immunoblotting with specific antibodies. Further, we demonstrated that co-expression of Zeb1 and CTBP2 in breast cancer patients correlated with the poor survival prognosis, thus signifying the functionality of the Zeb1–CTBP2 interaction.

Aim. To compare different methods of lentivirus concentration in order to select the best way of providing high-level transduction for generating laboratory CAR-T cells. Materials & Methods. Concentration of lentiviral supernatant was carried out by 4 methods: ultrafiltration, ultracentrifugation, polyethylene glycol (PEG), water-soluble non-ionic polymer, precipitation method, and ion-exchange chromatography. Functional viral titer was determined by mCherry reporter protein expression in the transduced HeLa cell line as well as by rapid immunochromatographic (IC) tests. Physical titer was determined by ELISA. Transduction efficiency of healthy donor’s T-lymphocytes was assessed by flow cytometry with respect to signal intensity of reporter protein FusionRed. Functional activity of generated anti-CD19 CAR-T was evaluated by microscopy after co-cultivation with CD19-HeLa cell line as well as subsequent cytokine testing. Results. Lentivirus purification and concentration by ultrafiltration provided the greatest number of transduced cells, i.e. 84.7 %. Methods of ultracentrifugation, PEG precipitation, and ion-exchange chromatography yielded 56.08 %, 74.22 %, and 21.05 % of T-cell transduction, respectively. Results of rapid IC tests were comparable (г = 0.91) with cell line titer data. The mean T-cell transduction efficiency was 59.55 % ± 2.94 %, and its maximum reached 76.26 %. Conclusion. The focus was laid on optimization of CAR-T cell production during the generation of lentiviral vectors and their purification. Ultrafiltration was selected as the best method of lentiviral supernatant concentration to efficiently transduce T-lymphocytes and to generate functional CAR-T cell population.

NK-cells as innate immunity elements manifest key reactions of antitumor immune response. NKG2D is an activating transmembrane receptor of NK-cells which is responsible for cytotoxicity initiation in response to the binding of specific ligands of genetically modified cells. Selective expression of NKG2D ligands provides a unique perspective on the therapy of wide variety of tumors. Acute myeloid leukemias (AML) are malignant hematological tumors with a high relapse risk. Due to the complexity of AML treatment strategy it is necessary to develop new approaches to tumor elimination using novel genetic constructs. Currently available CAR T-cell drugs with NKG2D receptor are successfully subjected to clinical studies in AML patients and prove their high therapeutic potential.

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