Fyn tyrosine kinase, a downstream target of receptor tyrosine kinases, modulates antiglioma immune responses

Abstract

Background High-grade gliomas are aggressive and immunosuppressive brain tumors. Molecular mechanisms that regulate the inhibitory immune tumor microenvironment (TME) and glioma progression remain poorly understood. Fyn tyrosine kinase is a downstream target of the oncogenic receptor tyrosine kinase pathway and is overexpressed in human gliomas. Fyn’s role in vivo in glioma growth remains unknown. We investigated whether Fyn regulates glioma initiation, growth and invasion. Methods We evaluated the role of Fyn using genetically engineered mouse glioma models (GEMMs). We also generated Fyn knockdown stem cells to induce gliomas in immune-competent and immune-deficient mice (nonobese diabetic severe combined immunodeficient gamma mice [NSG], CD8−/−, CD4−/−). We analyzed molecular mechanism by RNA sequencing and bioinformatics analysis. Flow cytometry was used to characterize immune cellular infiltrates in the Fyn knockdown glioma TME. Results We demonstrate that Fyn knockdown in diverse immune-competent GEMMs of glioma reduced tumor progression and significantly increased survival. Gene ontology (GO) analysis of differentially expressed genes in wild-type versus Fyn knockdown gliomas showed enrichment of GOs related to immune reactivity. However, in NSG and CD8−/− and CD4−/− immune-deficient mice, Fyn knockdown gliomas failed to show differences in survival. These data suggest that the expression of Fyn in glioma cells reduces antiglioma immune activation. Examination of glioma immune infiltrates by flow cytometry displayed reduction in the amount and activity of immune suppressive myeloid derived cells in the Fyn glioma TME. Conclusions Gliomas employ Fyn mediated mechanisms to enhance immune suppression and promote tumor progression. We propose that Fyn inhibition within glioma cells could improve the efficacy of antiglioma immunotherapies.

An Optimized Protocol for In Vivo Analysis of Tumor Cell Division in a Sleeping Beauty-Mediated Mouse Glioma Model

“…This workflow for inducing brain tumors in neonatal day 1 pups has been adopted from ( Wiesner et al, 2009 ) and is extensively used in our group to develop low or high grade glioma models harboring different genotypes ( Calinescu et al, 2015 ; Koschmann et al, 2016 ; Núñez et al, 2019 ; Garcia-Fabiani et al, 2020 ; Comba et al, 2020 ). Brain tumors are induced by transposon-mediated integration of genetic alterations delivered in plasmid DNA into the host genome, without the need of viral vectors ( Figures 1A and 1B ) ( Table 1 ).…”

“…In vivo profiling of glioma cells’ cell cycle analysis is performed in a genetically engineered glioma model in mice utilizing the Sleeping Beauty (SB) transposon system ( Wiesner et al, 2009 ; Calinescu et al, 2015 ; Koschmann et al, 2016 ; Núñez et al, 2019 ; Garcia-Fabiani et al, 2020 ; Comba et al, 2020 ). To achieve this, plasmids encoding the different genetic alterations of interest, which will be stably inserted into the genome of mouse brain cells, need to be constructed.…”

“…pKT-IRES-Katushka). Depending on the type of genetic alterations introduced, tumors will develop and animals will reach the humane endpoint within 2 to 6 months after the SB procedure ( Calinescu et al, 2015 ; Koschmann et al, 2016 ; Núñez et al, 2019 ; Comba et al, 2020 ) ( Table 1 ). In vivo 5-ethynyl-2′-deoxyuridine (EdU) staining is performed when the tumor is large, i.e., when bioluminescence reaches a signal of 10 6 photons/s/cm 2 /sr, but before the tumor burden endpoint.…”

Molecular Mechanisms of Treatment Resistance in Glioblastoma

“…MDSCs also secrete reactive oxygen and nitrogen species to induce apoptosis of natural killer and activated T cells, express IDO to deplete tryptophan and consequently impair cytotoxic T-cell responses, promote expansion of immunosuppressive T reg populations, and—importantly—promote immunosuppressive macrophage polarization by cell–cell contact [ 175 , 176 , 177 ]. A recent study has further elucidated the role of a non-receptor tyrosine kinase, Fyn, downstream of several crucial signaling pathways, including c-MET, EGFR, STAT3, and PIK3/Akt, not only in promoting the trafficking and expansion of MDSCs within the glioblastoma microenvironment, but also in promoting CD8+ T-cell exhaustion [ 178 ].…”

Caffeoyl-Prolyl-Histidine Amide Inhibits Fyn and Alleviates Atopic Dermatitis-Like Phenotypes via Suppression of NF-κB Activation

“…Fyn is a non-receptor tyrosine kinase belonging to the Src family kinases (SFKs), which include Blk, Brk, Fgr, Frk, Hck, Lck, Lyn, c-Src, Srm, and c-Yes in human [ 11 , 12 ]. Fyn transmits signals from diverse cell surface receptors to cytoplasmic signal transduction cascades [ 13 ]. It also plays important roles in the regulation of diverse biological functions, such as apoptosis, survival, adhesion, migration, neuronal transmission, and immunity [ 14 , 15 , 16 , 17 ].…”