Biology subjects

Grzywa, T. M.

Publications and source records attributed to Grzywa, T. M..

4 recordsLinked to original sources

bioRxiv2024

Enhanced lentiviral gene delivery to mammalian cells via paired cell surface and viral envelope engineering

Lentiviral vectors that facilitate gene delivery to desired cell types have been widely used in routine laboratory research and therapeutic cell engineering. However, the lack of proper entry receptors on many cell types often results in poor gene delivery. Here, we present a simple paired virus-cell engineering approach that promotes lentiviral gene delivery into mammalian cells. Lentiviruses are dual-pseudotyped with VSV-G and a chimeric envelope protein specifically recognizing a small molecule fluorescein (FITC-Env), and target cells are transiently labelled with FITC to create surrogate receptors for lentivirus attachment. The synthetic interaction between FITC-labeled cells and FITC-binding LVs enables efficient LV docking, viral entry and stable transgene expression in a range of mammalian cell lines and primary T cells. We showed that this approach enabled efficient delivery of a CD19-targeted chimeric antigen receptor (CAR) into naive human T cells that are naturally refractory to conventional VSV-G LVs, which upon activation rapidly eradicated CD19+ leukemic cells. This paired cell surface and virus envelope engineering approach may serve as a universal method for engineering synthetic virus-cell interactions to improve lentiviral gene delivery to mammalian cells.

bioengineering↗

bioRxiv2024

Estradiol induces global changes in miRNA expression in endometrial cancer cells and upregulates oncogenic miR-182

Endometrial cancer (EC) occurs mainly in perimenopausal age. Risk factors are associated with unopposed estrogen stimulation of the endometrium. There are two main types of EC, EC1 and EC2. The pathogenesis of EC1 is estrogen-dependent. MiRNAs are small RNAs that regulate gene expression posttranscriptionally. They are induced by estrogens in different hormone-dependent neoplasias including breast cancer. However, little is known regarding their role in EC. Thus, this study aims to assess the role of the miRNA-estrogen axis in endometrial cancer cells. Estrogen-dependent endometrial cancer cell line Ishikawa was used in the study. Cells were incubated with estradiol, followed by RNA isolation. We used the microarray method to identify estradiol-induced miRNAs in EC cells. Then, we analyzed tissues derived from 45 patients (18 EC1, 12 EC2, and 15 healthy endometrium (HE)) that were cut using the Laser Capture Microdissection method. The expression of selected miRNAs and their targets was assessed using the RT-qPCR method. Ishikawa cells were transfected with miRNA mimic, miRNA inhibitor (anti-miRNA), and their scrambled controls. We identified 66 estrogen-upregulated miRNAs in endometrial cancer cells. Out of them, miR-182 was upregulated in EC1 compared to HE. We found that miR-182 is an oncomiR in EC since its increased expression promoted the proliferation of EC cells and decreased expression of miR-182 was associated with the inhibition of cancer cell proliferation. Moreover, miR-182 inhibition upregulated SMAD4 expression. Our study allowed us to better understand the role of estrogen in the pathogenesis of EC.

cancer biology↗

bioRxiv2023

Ammonia inhibits antitumor activity of NK cells by decreasing mature perforin

Immunotherapy revolutionized cancer treatment in the last decade. Natural killer (NK) cells are one of the key host immunity components against malignant cells. Thus, they are currently extensively investigated in the field of immunotherapy of cancer. Different approaches have been developed to improve the antitumor activity of NK cells. Nonetheless, tumor microenvironment remains an obstacle to effective NK cell-based therapies. Here, we demonstrated that a cancer-conditioned medium suppresses the anti-tumor activity of NK cells. Further, we found that ammonia, a by-product of cancer cell metabolism, accumulates in the cancer-conditioned medium and tumor microenvironment. We identified that ammonia impairs the cytotoxicity of NK cells as well as the effectiveness of antibody-based and chimeric antigen receptor (CAR)-NK-based therapies in vitro. Inhibited activity of NK cells was caused by decreased levels of perforin. This effect was dependent on the lysosomotropic features of ammonia and its ability to increase pH in acidic compartments. In consequence, upon contact with ammonia the mature form of perforin was decreased in NK cells leading to their dysfunction. Our findings demonstrate that in addition to its previously described role of promoting tumor growth as a nitrogen source for tumor biomass ammonia could promote tumor escape as an NK cells immune checkpoint. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=66 SRC="FIGDIR/small/567708v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@19db6a5org.highwire.dtl.DTLVardef@1424903org.highwire.dtl.DTLVardef@c795d7org.highwire.dtl.DTLVardef@13e7a8e_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LICancer-conditioned medium suppresses the antitumor activity of NK cells C_LIO_LIAmmonia accumulates in conditioned medium and in the tumor microenvironment C_LIO_LIImpaired cytotoxicity of NK cells is caused by ammonia that decreases perforin levels C_LIO_LIAmmonia causes NK cell dysfunction C_LI

cancer biology↗

bioRxiv2021

Potent but transient immunosuppression of T-cells is a general feature of erythroid progenitor cells

CD71+ erythroid cells (CECs) have been recently recognized in both neonates and cancer patients as potent immunoregulatory cells. Here, we show that in mice early-stage CECs expand in anemia, have high levels of arginase 2 (ARG2) and reactive oxygen species (ROS). In the spleens of anemic mice, CECs expansion-induced L-arginine depletion suppresses T-cell responses. In humans with anemia, CECs expand and express ARG1 and ARG2 that suppress T-cells IFN-{gamma} production. Moreover, bone marrow CECs from healthy human donors suppress T-cells proliferation. CECs differentiated from peripheral blood mononuclear cells potently suppress T-cell activation, proliferation, and IFN-{gamma} production in an ARG- and ROS-dependent manner. These effects are the most prominent for early-stage CECs (CD71highCD235adim cells). The suppressive properties disappear during erythroid differentiation as more differentiated CECs and mature erythrocytes lack significant immunoregulatory properties. Our studies provide a novel insight into the role of CECs in the immune response regulation. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=179 SRC="FIGDIR/small/427109v2_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@136fbd8org.highwire.dtl.DTLVardef@d05d00org.highwire.dtl.DTLVardef@189baceorg.highwire.dtl.DTLVardef@1472b2b_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗