CIS deletion by CRISPR/Cas9 enhances human primary natural killer cell functions against allogeneic glioblastoma
Abstract Background Glioblastoma (GBM) is the most common malignant brain tumor and has “immunologically cold” features. Changing GBM to an “immunologically hot” tumor requires a strong trigger that induces initial immune responses in GBM. Allogeneic natural killer cells (NKCs) have gained considera...
| Main Authors: | , , , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
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BMC
2023-08-01
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| Series: | Journal of Experimental & Clinical Cancer Research |
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| Online Access: | https://doi.org/10.1186/s13046-023-02770-6 |
| _version_ | 1827632736582500352 |
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| author | Tsutomu Nakazawa Takayuki Morimoto Ryosuke Maeoka Ryosuke Matsuda Mitsutoshi Nakamura Fumihiko Nishimura Noriko Ouji Shuichi Yamada Ichiro Nakagawa Young Soo Park Toshihiro Ito Hiroyuki Nakase Takahiro Tsujimura |
| author_facet | Tsutomu Nakazawa Takayuki Morimoto Ryosuke Maeoka Ryosuke Matsuda Mitsutoshi Nakamura Fumihiko Nishimura Noriko Ouji Shuichi Yamada Ichiro Nakagawa Young Soo Park Toshihiro Ito Hiroyuki Nakase Takahiro Tsujimura |
| author_sort | Tsutomu Nakazawa |
| collection | DOAJ |
| description | Abstract Background Glioblastoma (GBM) is the most common malignant brain tumor and has “immunologically cold” features. Changing GBM to an “immunologically hot” tumor requires a strong trigger that induces initial immune responses in GBM. Allogeneic natural killer cells (NKCs) have gained considerable attention as promising immunotherapeutic tools against cancer, where gene-edited NKCs would result in effective anti-cancer treatment. The present study focused on the immune checkpoint molecule cytokine-inducible SH2-containing protein (CISH, or CIS) as a critical negative regulator in NKCs. Methods The GBM tumor environment featured with immunological aspect was analyzed with Cancer immunogram and GlioVis. We generated human primary CIS-deleted NKCs (NK dCIS) using clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) with single guide RNA targeting genome sites on CIS coding exons. The genome-edited NKCs underwent microarray with differential expression analysis and gene set enrichment analysis (GSEA). The anti-GBM activity of the genome-edited NKCs was evaluated by apoptosis induction effects against allogeneic GBM cells and spheroids. We further detected in vivo antitumor effects using xenograft brain tumor mice. Results We successfully induced human CIS-deleted NKCs (NK dCIS) by combining our specific human NKC expansion method available for clinical application and genome editing technology. CIS gene-specific guide RNA/Cas9 protein complex suppressed CIS expression in the expanded NKCs with high expansion efficacy. Comprehensive gene expression analysis demonstrated increased expression of 265 genes and decreased expression of 86 genes in the NK dCIS. Gene set enrichment analysis revealed that the enriched genes were involved in NKC effector functions. Functional analysis revealed that the NK dCIS had increased interferon (IFN)ɤ and tumor necrosis factor (TNF) production. CIS deletion enhanced NKC-mediated apoptosis induction against allogeneic GBM cells and spheroids. Intracranial administration of the allogeneic NKCs prolonged the overall survival of xenograft brain tumor mice. Furthermore, the NK dCIS extended the overall survival of the mice. Conclusion The findings demonstrated the successful induction of human primary NK dCIS with CRISPR/Cas9 with efficient expansion. CIS deletion enhanced the NKC-mediated anti-tumor effects in allogeneic GBM and could be a promising immunotherapeutic alternative for patients with GBM. |
| first_indexed | 2024-03-09T14:49:45Z |
| format | Article |
| id | doaj.art-dab32889ea614f1b8035bdd96dbc57d7 |
| institution | Directory Open Access Journal |
| issn | 1756-9966 |
| language | English |
| last_indexed | 2024-03-09T14:49:45Z |
| publishDate | 2023-08-01 |
| publisher | BMC |
| record_format | Article |
| series | Journal of Experimental & Clinical Cancer Research |
| spelling | doaj.art-dab32889ea614f1b8035bdd96dbc57d72023-11-26T14:34:38ZengBMCJournal of Experimental & Clinical Cancer Research1756-99662023-08-0142112210.1186/s13046-023-02770-6CIS deletion by CRISPR/Cas9 enhances human primary natural killer cell functions against allogeneic glioblastomaTsutomu Nakazawa0Takayuki Morimoto1Ryosuke Maeoka2Ryosuke Matsuda3Mitsutoshi Nakamura4Fumihiko Nishimura5Noriko Ouji6Shuichi Yamada7Ichiro Nakagawa8Young Soo Park9Toshihiro Ito10Hiroyuki Nakase11Takahiro Tsujimura12Grandsoul Research Institute for Immunology, IncDepartment of Neurosurgery, Nara Medical UniversityDepartment of Neurosurgery, Nara Medical UniversityDepartment of Neurosurgery, Nara Medical UniversityClinic Grandsoul NaraDepartment of Neurosurgery, Nara Medical UniversityDepartment of Immunology, Nara Medical UniversityDepartment of Neurosurgery, Nara Medical UniversityDepartment of Neurosurgery, Nara Medical UniversityDepartment of Neurosurgery, Nara Medical UniversityDepartment of Immunology, Nara Medical UniversityDepartment of Neurosurgery, Nara Medical UniversityGrandsoul Research Institute for Immunology, IncAbstract Background Glioblastoma (GBM) is the most common malignant brain tumor and has “immunologically cold” features. Changing GBM to an “immunologically hot” tumor requires a strong trigger that induces initial immune responses in GBM. Allogeneic natural killer cells (NKCs) have gained considerable attention as promising immunotherapeutic tools against cancer, where gene-edited NKCs would result in effective anti-cancer treatment. The present study focused on the immune checkpoint molecule cytokine-inducible SH2-containing protein (CISH, or CIS) as a critical negative regulator in NKCs. Methods The GBM tumor environment featured with immunological aspect was analyzed with Cancer immunogram and GlioVis. We generated human primary CIS-deleted NKCs (NK dCIS) using clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) with single guide RNA targeting genome sites on CIS coding exons. The genome-edited NKCs underwent microarray with differential expression analysis and gene set enrichment analysis (GSEA). The anti-GBM activity of the genome-edited NKCs was evaluated by apoptosis induction effects against allogeneic GBM cells and spheroids. We further detected in vivo antitumor effects using xenograft brain tumor mice. Results We successfully induced human CIS-deleted NKCs (NK dCIS) by combining our specific human NKC expansion method available for clinical application and genome editing technology. CIS gene-specific guide RNA/Cas9 protein complex suppressed CIS expression in the expanded NKCs with high expansion efficacy. Comprehensive gene expression analysis demonstrated increased expression of 265 genes and decreased expression of 86 genes in the NK dCIS. Gene set enrichment analysis revealed that the enriched genes were involved in NKC effector functions. Functional analysis revealed that the NK dCIS had increased interferon (IFN)ɤ and tumor necrosis factor (TNF) production. CIS deletion enhanced NKC-mediated apoptosis induction against allogeneic GBM cells and spheroids. Intracranial administration of the allogeneic NKCs prolonged the overall survival of xenograft brain tumor mice. Furthermore, the NK dCIS extended the overall survival of the mice. Conclusion The findings demonstrated the successful induction of human primary NK dCIS with CRISPR/Cas9 with efficient expansion. CIS deletion enhanced the NKC-mediated anti-tumor effects in allogeneic GBM and could be a promising immunotherapeutic alternative for patients with GBM.https://doi.org/10.1186/s13046-023-02770-6Natural killer cellGlioblastomaCRISPR/Cas9CISPeripheral blood |
| spellingShingle | Tsutomu Nakazawa Takayuki Morimoto Ryosuke Maeoka Ryosuke Matsuda Mitsutoshi Nakamura Fumihiko Nishimura Noriko Ouji Shuichi Yamada Ichiro Nakagawa Young Soo Park Toshihiro Ito Hiroyuki Nakase Takahiro Tsujimura CIS deletion by CRISPR/Cas9 enhances human primary natural killer cell functions against allogeneic glioblastoma Journal of Experimental & Clinical Cancer Research Natural killer cell Glioblastoma CRISPR/Cas9 CIS Peripheral blood |
| title | CIS deletion by CRISPR/Cas9 enhances human primary natural killer cell functions against allogeneic glioblastoma |
| title_full | CIS deletion by CRISPR/Cas9 enhances human primary natural killer cell functions against allogeneic glioblastoma |
| title_fullStr | CIS deletion by CRISPR/Cas9 enhances human primary natural killer cell functions against allogeneic glioblastoma |
| title_full_unstemmed | CIS deletion by CRISPR/Cas9 enhances human primary natural killer cell functions against allogeneic glioblastoma |
| title_short | CIS deletion by CRISPR/Cas9 enhances human primary natural killer cell functions against allogeneic glioblastoma |
| title_sort | cis deletion by crispr cas9 enhances human primary natural killer cell functions against allogeneic glioblastoma |
| topic | Natural killer cell Glioblastoma CRISPR/Cas9 CIS Peripheral blood |
| url | https://doi.org/10.1186/s13046-023-02770-6 |
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