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Michael Platten et al.
of Heidelberg Cancer Research Center in Germany defined the myeloid state in IDH mutant glioma by longitudinal single-cell atlas and demonstrated that it is tightly controlled by tumor genotype, an article published in the July
2021 issue of Nature Cancer.
—Excerpted from the article chapter
【Ref: Friedrich M, et al.
Nat Cancer.
2021 Jul;(2)723-740.
doi: 10.
1038/s43018-021-OO201-z.
】
Research background
The glioma microenvironment is closely related to
tumor evolution, progression, and treatment resistance.
In high-grade gliomas (HGGs), microglia and monocyte-derived macrophages are collectively referred to as glioma-associated myeloid cells (GAM), which make up 70%
of tumors.
The current view is that hematogenous macrophages are recruited into the glioma microenvironment, in which the phenotype and functional modeling of invading macrophages and resident microglia depend on the genotype of the tumor, such as mutations encoding IDH1 type (IDH1) genes, which have a causal relationship with the intrinsic epigenetic and metabolic changes of tumor cells, and are also associated
with a good prognosis of glioma patients.
Functionally altered GAM in turn promotes tumor growth
through various mechanisms.
A distinctive feature of GAM is antigen presentation ability and acquisition of immunosuppressive phenotypes
.
In myeloid cells, microglia-specific versus macrophage-specific genes are continuously distributed, rather than bimodal distribution
.
The temporal cell-specific function of the glioma microenvironment is not well understood
.
Michael Platten et al.
of Heidelberg Cancer Research Center in Germany defined the myeloid state in IDH mutant glioma by longitudinal single-cell atlas and demonstrated that it is tightly controlled by tumor genotype, an article published in the July
2021 issue of Nature Cancer.
Research methods
on 30,000-600,000 microglia and macrophages screened from 14 HGGs.
Principal component analysis showed that CD45+CD3−CD19−CD20− were isolated from 5 cases of IDH wild type (WT) and 5 cases of IDH mutant HGG based on the mutation state of IDH Hematopoietic cells were subjected to single-cell RNA-seq (scRNA-seq) and compared with 7 brain tissue controls, and 10 clusters with different transcriptions were found, corresponding to different cell types and states
.
The authors used hypergeometric tests to find that myeloid cell clusters C:C0, C3, C4, and C6 were enriched in control tissue
.
Microglia homeostasis genes (TMEM119, P2RY12, CSF1R) are downregulated, while interferon (IFN) signaling pathways and hypoxia-related genes are upregulated
.
Differential gene expression analysis of microglia and macrophage clusters C0-C6 showed upregulation
of major histocompatibility complex (MHC) class I and II coding genes, including HLA-B, CD74, and HLA-DPA1.
Compared to IDH-WT HGGG-associated GAM, the latter showed upregulation
of homeostatic microglia and inflammatory mediator-coding genes such as P2RY12 and IL1B.
Next, the researchers used time-of-flight technology (CyTOF) to validate the scRNA-seq findings
at the protein level.
According to transcriptome analysis, the downregulation of myeloid cell microglia homeostatic signal derived from IDH-mutant HGG was not obvious, and the upregulation of AP signal was not obvious
, compared with IDH-WT type HGG.
Similar differences
can be detected using different combinations of CyTOF antibodies.
Overall, the above data suggest that in human HGG, the genotype dependence of GAM forms a significant and differentiated immunosuppressive phenotype
.
To investigate the dynamics and underlying molecular mechanisms of this glioma genotype-dependent GAM immunosuppressive phenotype, the authors used a mouse model
of GL261 overexpressing WT and mutant IDH HGG.
The authors then performed scRNA-seq on CD45+ cells purified from flow cytometry isolated from IDH mutants and IDH-WT GL261 gliomas, including microglia, monocytes, macrophages, and monocyte-derived dendritic cells (DCs), mast cells, granulocytes, and T and B cells
.
Flow cytometry
is performed at two time points of glioma progression, i.
e.
, day 7 and day 28 after primary tumor.
The results showed that on day 7, the microglia were made up
of more than 75% myeloid cell tumors.
On day 28, however, granulocytes predominate
.
At day 7, aggressive immune cells were more abundant in IDH-WT compared to IDH-mutant glioma, while in advanced stages, hematopoietic immune cells were comparable
in both experimental HGGs.
There are large numbers of circulating immune cells
in the early and late periods.
The authors hypothesize that, depending on their IDH status, microglia shaped by the early HGG microenvironment drive the differentiation of immune cells, particularly bloodborne macrophages
.
In early experimental HGG, differential expression analysis of microglia further showed that the gene expression of IDH-WT glioma-encoding MHC and co-stimulatory molecules was increased, while IDH-mutant glioma cells showed higher steady-state microglial gene expression
.
To assess the gradual change between homeostasis and activation of microglia, a pseudo-temporal analysis of early microglia using StemID2 showed a transition
from a steady-state state to a differentiated state.
In conclusion, IDH-mutant HGG showed decreased transcription profiles of early immunogenic microglia, decreased content of invasive myeloid cells, and increased
levels during tumor progression.
The authors studied the functional phenotype of cells derived from monocytes at late time points and found strong expression
of AP characteristics in both cells.
DCs exhibit only moderate features in IDH mutant types compared to IDH-WT tumors, while macrophages of IDH-mutant gliomas show upregulation of Il1b and downregulation
of Arg1.
In vitro co-culture studies, observing primitive T cells, microglia and macrophages isolated from experimental HGGs found that in cytotoxic cells and T helper cells, the production of IFN-γ was consistent with the upregulation of programmed cell death protein (PD)-1 and was proportionally dependent, and the production of granzyme (Grz) B in cytotoxic T cells was reduced
.
There are differences
in the level of T cell inhibition in macrophages other than microglia in the IDH mutation state.
To clarify this time-dependent and functional transformation molecular mechanism of tumor genotype, the authors exposed human monocytes and macrophages to a new form enzymatic product of the IDH mutant, R-2-hydroxyglutaric acid (R-2-HG).
Co-incubation of R-2-HG pretreated monocytes or macrophages with T cells found that T cell proliferation was dose-dependently inhibited
.
A dose-dependent downward regulation
of these proteins was observed after R-2-HG exposure.
Macrophages and microglia receive exogenous R-2-HG
independently of the activated state.
Overexpression of amino acid transporters known to transport R-2-HG, such as solute carrier (SLC) 13A3, can lead to increased
uptake of R-2-HG.
In order to verify the specific expression of AHR target gene in GAM, the authors performed pseudo-temporal trajectory analysis of human glioma infiltration, and found that the AHR target gene was upregulated in IDH mutant GAM, but not in IDH wild-type GAM
.
According to the human dataset, all clustered cells that formed myeloid cell trajectories in experimental HGG showed differences
in the cumulative expression of AHR-activating genes between IDH mutant type cells and IDH wild-type cells.
AHR transcripts are most abundant in monocytes, indicating that immune cell isotype-specific is susceptible to reprogramming
by R-2-HG.
AHR has been identified as a key cofactor for immunosuppressively transforming growth factor (TGF)-β and interleukin (IL)-1β signal transduction; AHR directly contributes to IL-10 production
.
Research results
Deletion of L-tryptophan (L-Trp) has been shown to reduce the translocation
of R-2-HG to AHR.
Cumulative data suggest that R-2-HG is not a direct ligand for AHR, and to assess R-2-HG-mediated inhibition of T cells by macrophages dependent on L-Trp, the authors performed co-culture assays
with macrophages in L-Trp-free and control media, respectively.
Deprivation of tryptophan from macrophages exposed to R-2-HG can lead to increased
effector function of co-incubated T cells.
Based on these findings, the authors hypothesized that immunosuppressive L-Trp catabolism via the kynurenine pathway drives macrophage infiltration in the reprogramming
of IDH-mutant tumors.
The rate-limiting step catalyzing the canine purine pathway by tryptophan 2,3-dioxygenase (TDO2) and indolamine 2,3-dioxygenase (IDO)1 and IDO2 together account for 90%
of L-Trp degradation.
Ratios of kynurenine-to-tryptophan ([L-Kyn]/[L-Trp]) in plasma are often used to express or reflect the activity
of these enzymes.
To determine the mechanism of L-Trp degradation in macrophages, the authors performed cell-free enzyme analysis
with rate-limiting enzymes in the kynurenine pathway.
The authors found that in macrophages, TDO2 is directly induced by R-2-HG, leading to the accumulation
of the AHR ligand L-Kyn.
Because for animal cells, de novo synthesis of L-Trp is impossible
.
Exposure of human monocyte-derived macrophages to R-2-HG results in an amino acid transporter expression pattern similar to extracellular L-Trp deletion, indicating that R-2-HG leads to increased L-Trp degradation in macrophages, resulting in an amino acid starvation-like response
.
Compared with IDH-WT tumors, the difference in IDH mutant type is upregulated and shows a consistent expression pattern
on the monocyte-macrophage trajectory.
The results suggest that transmembrane transport of branched-chain amino acids such as L-Trp is preferentially mediated by LAT1-CD9829 and therefore may provide the L-Trp
required for R-2-HG to maintain activation of the kynurenine pathway.
The authors studied the inhibition of LAT1-CD98 in vivo and in vitro
.
Pretreatment of monocyte-derived macrophages with small molecule LAT1-CD98 inhibitors rescues 17 of the 22 AHR targets induced by R-2-HG
.
Similarly, the use of LAT1-CD98 inhibitors in animal models of gliomas resulted in an increase in the number of MHCII+CD80+CD86+ immune-stimulating macrophages in IDH-mutant tumors, enhancing AP signaling
.
Macrophages exposed to R-2-HG and deprivation of tryptophan leads to enhanced
effector function of co-incubated T cells.
The authors' study aimed to determine whether the microenvironment of human IDH-mutant gliomas has the effect
of maintaining the L-tryptophan-dependent axis.
Using a matrix-assisted laser desorption-ionization (MALDI)-MS imaging (MSI)-based analytical method, it was found that in human HGG tissues, L-Trp aggregated outside the cell, and as expected, all IDH mutant tumors showed accumulation of R-2-HG, while R-2-HG
was not detected in either IDH wild-type HGG or control samples.
There is a large accumulation of L-Trp outside the cells of IDH-mutant HGG, which is significantly higher than that of IDH-WT HGG
.
The L-Trp level in IDH-WT HGG was moderate, and the L-Trp level was significantly lower than that in the tumor sample
.
The authors also found that intracellular and extracellular L-Trp levels of IDH-mutant HGG were moderately high and elevated, respectively; Macrophages exhibit moderate levels of TDO2 expression, and in IDH-mutant tumors, TDO2 expression levels are not increased
.
Overall, myeloid cells maintain immunosuppressive reprogramming
of GAM in IDH-mutant HGG through overstimulation of L-trp by LAT1-CD98 and R-2-HG-dependent uptake.
Conclusion of the study
of L-Trp and AHR functions.
The AHR target screening array showed that inhibition of AHR or LAT1-CD98 by small molecule inhibitors was effective in restoring R-2-HG-mediated macrophage reprogramming
.
When co-cultured with AHR-deficient macrophages exposed to R-2-HG, there was no same inhibition
of T cell proliferation or effector function.
In IDH-mutant tumors, AHR inhibition can reduce the production of IL-10 and TGF-β derived from monocytes, which is not the case in
IDH-WT tumors.
Finally, the authors note that there is a genotype-dependent intratumor-based network in glioma that consists of myeloid cells and determine that tryptophan metabolism is the target of
immunotherapy for IDH-mutant tumors.
