Dual-sgRNA CRISPR/Cas9 knockout of PD-L1 in human U87 glioblastoma tumor cells inhibits proliferation, invasion, and tumor-associated macrophage polarization

Abstract

Programmed death ligand 1 (PD-L1) plays a key role in glioblastoma multiforme (GBM) immunosuppression, vitality, proliferation, and migration, and is therefore a promising target for treating GBM. CRISPR/Cas9-mediated genomic editing can delete both cell surface and intracellular PD-L1. This systemic deliverable genomic PD-L1 deletion system can be used as an effective anti-GBM therapy by inhibiting tumor growth and migration, and overcoming immunosuppression. To target PD-L1 for CRISPR/Cas9 gene editing, we first identified two single guide RNA (sgRNA) sequences located on PD-L1 exon 3. The first sgRNA recognizes the forward strand of human PD-L1 near the beginning of exon 3 that allows editing by Cas9 at approximately base pair 82 (g82). The second sgRNA recognizes the forward strand of exon 3 that directs cutting at base pair 165 (g165). A homology-directed repair template (HDR) combined with the dual-sgRNAs was used to improve PD-L1 knockout specificity and efficiency. sgRNAs g82 and g165 were cloned into the multiplex CRISPR/Cas9 assembly system and co-transfected with the HDR template in human U87 GBM cells (g82/165 + HDR). T7E1 analysis suggests that the dual-sgRNA CRISPR/Cas9 strategy with a repair template was capable of editing the genomic level of PD-L1. This was further confirmed by examining PD-L1 protein levels by western blot and immunofluorescence assays. Western blot analysis showed that the dual-sgRNAs with the repair template caused a 64% reduction of PD-L1 protein levels in U87 cells, while immunostaining showed a significant reduction of intracellular PD-L1. PD-L1 deletion inhibited proliferation, growth, invasion and migration of U87 cells, indicating intracellular PD-L1 is necessary for tumor progression. Importantly, U87 cells treated with g82/165 + HDR polarized tumor-associated macrophages (TAM) toward an M1 phenotype, as indicated by an increase in TNF-α and a decrease in IL-4 secretions. This was further confirmed with flow cytometry that showed an increase in the M1 markers Ly6C + and CD80 +, and a decrease in the M2 marker CD206 + both in vitro and in vivo. Utilizing dual-sgRNAs and an HDR template with the CRISPR/Cas9 gene-editing system is a promising avenue for the treatment of GBM.

Novel macrophage-related gene prognostic index for glioblastoma associated with M2 macrophages and T cell dysfunction

“…TAMs are responsible for the production of such inflammatory response mediators in the TME ( 39 ). Notably, TAMs are involved in the dysfunction of CD8 T cells by expressing multiple immune checkpoints and the induction of other cells in the TME in expressing immune checkpoints ( 13 , 15 ). On this basis, we consider that the construction of gene signatures associated with TAMs may also be useful for immunotherapy.…”

“…Tumor-associated macrophages (TAMs) in GBM refer to blood-derived monocytes/macrophages and intrinsic microglia, and the alternatively activated TAMs orchestrate an immunosuppressive TME thus impeding the anti-tumor immune activity ( 10 – 12 ). Interstingly, increasing evidence suggests that TAMs involve in the expression of immune checkpoints in the TME and play a vital role in inducing CD8 T lymphocyte dysfunction ( 11 , 13 – 15 ). Given that GBM is a class of TAMs-rich tumors ( 16 ), the identification of biomarkers associated with alternative activation of TAMs may provide prognostic convenience for GBM on the one hand, and pave the way for the application of ICI on the other.…”

Origin, activation, and targeted therapy of glioma-associated macrophages

“…These small molecule inhibitors sensitized tumors to the immune checkpoint inhibitor combination of anti-PD1 plus anti-CTLA4 antibodies and prolonged the survival of animals in two syngeneic GBM models ( 161 ). With the development of gene-editing technologies, CRISPR/Cas9-mediated PD-L1 knockout using dual single-guide RNAs (sgRNAs) and homology-directed repair (HDR) template is also a promising therapeutic option ( 162 ).…”

Genome editing

“…Application of PITCh (Precise Integration into Target Chromosome) 45 to target the Ca2 + sensor GCaMP3 into the midge Polypedilum vanderplanki Pv11 cells suggests an important role for calcium signaling in anhydrobiosis in midge larvae 40 . HDR has also been used to increase the robustness of PD-L1 gene knock-down in cultured glioblastoma cells, by introducing a template harboring a stop codon, revealing a critical role for PD-L1 in preventing proliferation, invasion and tumor associated macrophage polarization to M1 phenotype 39 .…”

“…These powerful Cas9-based genome editing tools allow targeted modification of nearly any gene of interest across most biological systems, enabling molecular genetic studies in organisms in which gene manipulation and transgenesis has traditionally been difficult 25,26 . Key considerations in the application of this technology are optimal delivery and expression of CRISPR/Cas9 reagents.This Collection gathers 17 contributions describing new methods to improve CRISPR/Cas9 genome editing efficiency in a variety of animal and plant systems [27][28][29][30][31][32][33][34] , and its application to develop new genetic tools and models [35][36][37][38] and to gain novel insight into mechanisms regulating organismal physiology, cell behavior, and gene expression [39][40][41][42][43] .The relatively low efficiency of CRISPR/Cas9 targeted integration of exogenous DNA via HDR has been a limiting factor in precision knock-in in mammalian cells and in vivo systems in which the predominant DNA repair pathway is Non-Homologous End Joining (NHEJ). Optimization of each parameter in CRISPR knock-in experimental design, including the specific Cas endonuclease, single or double-stranded DNA template, linear or circular templates, and homology arm length increases the chances of recovering precision HDR edits.…”