Genetic engineered primary human natural killer cells targeting TIGIT via CRISPR/Cas9 enhanced anti-tumor activity against GBM spheroids and 2D culture.
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ABSTRACT:
PROVIDER: PRJNA878498 | ENA |
REPOSITORIES: ENA
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Project description:Background. Although several immunotherapies against glioblastoma (GBM) have been investigated for long time, only limited effective results are acquired. Therefore, we developed immunotherapy based on genome edited NK cells knocking out the checkpoint receptor, which would overcome the immunosuppressive tumor microenvironment in GBM. Methods. We generated T cell immunoglobulin and ITIM domain (TIGIT), an inhibitory receptor expressed on lymphocytes, knockout (KO) human primary NK cells using the clustered regularly interspaced short palindromic repeats (CRISPR)/ CRISPR-associated protein9 (Cas9), each single guide RNA targeting different genome sites on TIGIT coding exons. To detect anti-tumor activity of genome edited NK cells against GBM, we utilized 2D adherent model and spheroids derived from GBM cell lines, U87, T98G, LN18, and U251. Subsequently, we performed real time cell growth assays, flow cytometry based apoptosis assays, and ELISA for investigating anti-tumor activity. Result. We established TIGIT KO human primary NK cells using CRISPR/Cas9. Flow cytometry indicated effective knockout of TIGIT and unchanged expression of immune checkpoint receptors other than TIGIT. T7 endonuclease I mutation detection assays showed that RNPs disrupted the intended genome sites. Using real time cell growth assays, we revealed enhanced anti-tumor activity of genome edited NK cells against 2D adherent GBM cells. The genome edited NK cells also exhibits enhanced anti-tumor effect against GBM spheroids derived from GBM cell lines.Conclusion. Our founding suggests that immunotherapy based on TIGIT KO NK cells using CRISPR/Cas9 is a promising therapy against GBM.
2022-09-11 | GSE212941 | GEO
Project description:GBM is the most common and aggressive primary brain tumor with dismal prognosis. NK cells are large granular lymphocytes with natural cytotoxicity against tumor cells, and they should be established for the novel treatment against patients with GBM. We have previously reported highly activated and ex vivo-expanded NK cells derived from human peripheral blood, which is designated as Genuine-induced natural killer cell (GiNK), induced by our specific culture conditions exerted the cytotoxic effect on GBM cells via apoptosis. First, we comprehensively summarized the molecular characteristics, especially focusing on the expression of stem cell markers, extracellular matrix markers, chemokine, chemokine receptors, and the NK receptors’ ligands, of GBM spheroids compared with 2D adherent GBM cells by using microarray. In the spheroid derived from GBM cell lines, gene expression of stem cell markers, extracellular matrix markers, chemokine, chemokine receptor, NK cell inhibitory receptor’ ligand were up-regulated compared with 2D adherent GBM cells. A preclinical evaluation of the NK cells was performed by ex vivo 3D spheroid model derived from GBM cell lines. In 3D spheroid model, the NK cells accumulated and infiltrated around the spheroids and induced the GBM cell death. Flow cytometry-based apoptosis detection assay clearly showed that the NK cells induced the GBM cell death via apoptosis. Our findings could provide pivotal information for the NK cell based-immunotherapy to patients with GBM.
2021-08-19 | GSE182373 | GEO
Project description:GBM is the most common and aggressive primary brain tumor with dismal prognosis. NK cells are large granular lymphocytes with natural cytotoxicity against tumor cells, and they should be established for the novel treatment against patients with GBM. We have previously reported highly activated and ex vivo-expanded NK cells derived from human peripheral blood, which is designated as Genuine-induced natural killer cell (GiNK), induced by our specific culture conditions exerted the cytotoxic effect on GBM cells via apoptosis. First, we comprehensively summarized the molecular characteristics, especially focusing on the expression of stem cell markers, extracellular matrix markers, chemokine, chemokine receptors, and the NK receptors’ ligands, of GBM spheroids compared with 2D adherent GBM cells by using microarray. In the spheroid derived from GBM cell lines, gene expression of stem cell markers, extracellular matrix markers, chemokine, chemokine receptor, NK cell inhibitory receptor’ ligand were up-regulated compared with 2D adherent GBM cells. A preclinical evaluation of the NK cells was performed by ex vivo 3D spheroid model derived from GBM cell lines. In 3D spheroid model, the NK cells accumulated and infiltrated around the spheroids and induced the GBM cell death. Flow cytometry-based apoptosis detection assay clearly showed that the NK cells induced the GBM cell death via apoptosis. Our findings could provide pivotal information for the NK cell based-immunotherapy to patients with GBM.
2021-08-19 | GSE182374 | GEO
Project description:TIGIT+ Tregs suppress Th1 and Th17 responses while sparing Th2 responses. Analysis of global gene expression of TIGIT+ vs. TIGIT- Tregs from naive mice reveled that TIGIT+ Tregs display an activated phenotype and are enriched for Treg signature genes including the Treg effector molecule Fgl2 which enables them to selectively spare Th2 responses. TIGIT+ and TIGIT- Tregs were sorted from naïve Foxp3-GFP KI mice (pooled spleen and lymph nodes) TIGIT: T cell immunoreceptor with Ig and ITIM domains
2014-12-12 | E-GEOD-56299 | biostudies-arrayexpress
Project description:Using the multi-inlet spheroid generator (MSG) platform, we generated stable GBM spheroids and observed their interactions with various immune cells, including microglia and natural killer (NK) cells, at different time points. HMO6 and NK-92 cells were used for microglia and NK cells, respectively. We examined the interactions between tumor-immune cells as well as between different immune cells. The generation of tri-cultured spheroids successfully recapitulated the immunosuppressive microenvironment of the GBM tumor.
2024-07-06 | GSE271170 | GEO
Project description:Human bone marrow mesenchymal stromal cells (MSCs) are conventionally cultured as adherent monolayers on tissue culture plastic. MSCs can also be cultured as 3D cell aggregates (spheroids). Optimised 3D conditions (60,000 MSCs cultured as a spheroid for 5 days) inhibited MSC proliferation and induced cell shrinkage in the absence of cell death. Primary human MSCs isolated from 2 donors were cultured under both monolayer (2D MSCs) and optimised 3D (3D MSCs) conditions. High quality RNA was isolated from all samples, and global gene expression analysis was performed in duplicate (using Agilent SurePrint G3 Human Gene Expression 8x60K v2 Microarrays) to identify gene expression changes in 3D compared to 2D MSC cultures.
2015-09-30 | E-MTAB-3795 | biostudies-arrayexpress
Project description:TIGIT+ Tregs suppress Th1 and Th17 responses while sparing Th2 responses. Analysis of global gene expression of TIGIT+ vs. TIGIT- Tregs from naive mice reveled that TIGIT+ Tregs display an activated phenotype and are enriched for Treg signature genes including the Treg effector molecule Fgl2 which enables them to selectively spare Th2 responses.
2014-12-12 | GSE56299 | GEO
Project description:Spheroids are 3D multi-cell aggregates formed in non-addherent culture conditions. In ovarian cancer (OC), they serve as a vehicle for cancer cell dissemination in the peritoneal cavity. We investigated genes and networks upregulated in three dimensional (3D) versus two-dimensional (2D) culture conditions by Affymetrix gene expression profiling and identified ALDH1A1, a cancer stem cell marker as being upregulated in OC spheroids. Network analysis confirmed ALDH1A1 upregulation in spheroids in direct connection with elements of the beta-catenin pathway. A parallel increase in the expression levels of beta-catenin and ALDH1A1 was demonstrated in spheroids vs. monolayers an in successive spheroid generations by using OC cell liness and primary OC cells. The percentage of Aldefluor positive cells was significantly higher in spheroids vs. monolayers in IGROV1, A2780, SKOV3, and primary OC cells. B-catenin knock-down decreased ALDH1A1 expression and chromatin immunoprecipitation demonstrated that beta-catenin directly binds to the ALDH1A1 promoter. Both siRNA mediated beta-catenin knock-down and a novel ALDH1A1 small molecule enzymatic inhibitor described here for the first time, decreased the number of OC spheroids (p<0.001) and cell viability. These data strongly support the role of beta-catenin regulated ALDH1A1 in the maintenance of OC spheroids and of a stem cell phenotype and propose new ALDH1A1 inhibitors targeting this cell population. Different gene profiles were observed in ovarian cancer spheroids versus ovarian cancer monolayers. Nine samples were analyzed in triplicate. Each group is a reference.
2014-01-03 | E-GEOD-53759 | biostudies-arrayexpress
Project description:We compared the gene expression in wild type CD4+ T cells stimulated with agonistic anti-TIGIT 4D4 antibody to that of isotype and TIGIT-deficient (KO) controls.
2011-01-01 | E-MEXP-2847 | biostudies-arrayexpress
Project description:Liver-derived cells from the surface and cave-adapted morphs of Astyanax mexicanus are valuable in vitro resources to explore the metabolism of these unique fish. However, 2D cultures have not yet fully mimicked the metabolic profile of the fish liver. Also, 3D culturing can modulate the transcriptomic profile of cells when compared to its 2D counterpart. Hence, to widen the range of metabolic pathways that can be depicted in vitro, we cultured the liver-derived SFL and CFL into 3D spheroids. We 3D cultured the cells at various cell seeding densities for 4 weeks and characterized the resultant transcriptome. The 3D cultured SFL and CFL cells indeed depicted a wider range of metabolic pathways as compared to the 2D culture. Further, the 3D spheroids also showed surface and cave-specific responses, making the spheroids an exciting system to study cave adaptation. Taken together, SFL and CFL spheroids prove to be a promising model for overall understanding of altered metabolism in Astyanax mexicanus.
2023-06-13 | GSE234212 | GEO