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Glioma-induced inhibition of caspase-3 in microglia promotes a tumor-supportive phenotype

Abstract

Glioma cells recruit and exploit microglia (the resident immune cells of the brain) for their proliferation and invasion ability. The underlying molecular mechanism used by glioma cells to transform microglia into a tumor-supporting phenotype has remained elusive. We found that glioma-induced microglia conversion was coupled to a reduction in the basal activity of microglial caspase-3 and increased S-nitrosylation of mitochondria-associated caspase-3 through inhibition of thioredoxin-2 activity, and that inhibition of caspase-3 regulated microglial tumor-supporting function. Furthermore, we identified the activity of nitric oxide synthase 2 (NOS2, also known as iNOS) originating from the glioma cells as a driving stimulus in the control of microglial caspase-3 activity. Repression of glioma NOS2 expression in vivo led to a reduction in both microglia recruitment and tumor expansion, whereas depletion of microglial caspase-3 gene promoted tumor growth. Our results provide evidence that inhibition of the denitrosylation of S-nitrosylated procaspase-3 mediated by the redox protein Trx2 is a part of the microglial pro-tumoral activation pathway initiated by glioma cancer cells.

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Figure 1: Glioma cells promote diminished basal caspase-3 activity in microglia cells.
Figure 2: Knockdown of caspase-3 promotes the microglial tumor-supportive phenotype.
Figure 3: C6 glioma cells counteract LPS-induced DEVDase activity and NOS2 expression in BV2 microglia cells.
Figure 4: Glioma NOS2 contributes to S-nitrosylation of microglial caspase-3.
Figure 5: Inhibition of microglial Trx2 activity promotes S-nitrosylation of mitochondrial caspase-3.
Figure 6: Inhibition of glioma NOS2 restricts inhibition of microglial caspase-3 activity, microglia recruitment and tumor growth.
Figure 7: Depletion of microglial procaspase-3 promotes glioma tumor growth.

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Acknowledgements

We thank G. Brown (University of Cambridge) for the BV2 cell line; R. Glass (Max Delbruck Center) for the GL261 cell line; O. Hermanson (Karolinska Institutet) for the C6 cell line; M. Nister (Karolinska Institutet) for the U-251MG, U-343MG, U-373MG, U-87MG and U-1241MG cell lines; M. Schultzberg (Karolinska Institutet) for the CHME3 cell line; and the CLICK Imaging Facility (supported by the Knut and Alice Wallenberg Foundation) for technical support. Supported by the Karolinska Institutet Foundation (X.S. and B.J.), the Swedish Childhood Cancer Foundation (A.M.O., B.J. and K.B.), the Swedish Research Council (M.A.B. and B.J.), the Strategic Research Programme in Cancer (B.J.), the Strategic Research Programme in Neuroscience (K.B.), the Swedish Cancer Foundation (B.J.), Spanish MINECO/FEDER/UE (J.L.V.), the Swedish Cancer Society (B.J.), the Swedish Brain Foundation (B.J.) and Swedish governmental grants for researchers working in healthcare (K.B.).

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Authors and Affiliations

Authors

Contributions

X.S. and M.A.B. performed all of the experiments except otherwise noted; A.M.O., A.C.-J., J.L.V. and K.B. contributed with in vivo analyses; V.R., U.N. and J.H. participated with the human xenograft mouse model. J.F. contributed with the biotin switch method analysis; M.A. and A.Ö. generated the shRNA NOS2 stable transfectant; S.K. and A.B. contributed with primary microglial cell culture preparation; A.R. and R.A.F. provided the Casp3flox/flox mice; D.S. and J.R. participated with some of the coculture experiments; E.K. was involved in study design; X.S., M.A.B. and B.J. designed the study, and analyzed and interpreted the data; M.A.B. and B.J. wrote the first draft of the manuscript; and all authors discussed the results and commented on or edited the manuscript.

Corresponding author

Correspondence to Bertrand Joseph.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 In vitro and in vivo model systems used in these studies and measurement of in vitro caspase-8 activity.

(a) Diagram illustrating a transwell segregated microglia-glioma coculture setup to study glioma’s influence over microglia biology. (b) Measurement of LETDase (caspase-8) activity in BV2 microglia cells cultured in medium alone or segregated coculture with a panel of glioma cells at 6h (black bars) and 24h (white bars) after coculture. Results are presented relative to those of BV2 microglia cultured in medium alone, set as 1. (c) Illustration of how postnatal day 16-17 male C57/BL6/J mice were injected intrastriatally with syngeneic GFP-expressing GL261 glioma cells. One or two weeks after glioma injection, mice were sacrificed and brain tissue processed for analysis. Statistics and error bars: mean ± s.d. n=6 (b) of biological replicates. Data was analyzed using two-tailed Student’s t-test. ns, not significant (P > 0.05); *P< 0.05; **P< 0.01.

Supplementary Figure 2 Microglial active caspase-3 expression in intracranial human glioblastoma xenografts.

(a, b) Representative pictures of the immunohistochemical analysis of cleaved caspase-3 (red) in Iba-1 positive cells (microglia; white) inside (a) and in the border (b) of tumors formed 1 week after transplantation of human U-87MG glioblastoma cells (green) into NOD.CB17-PrkcSCID/J mice brains (n=6/group).

Supplementary Figure 3 Effect of the silencing of microglial caspase-3 on the expression of Il6, Ccl22, Chil3, Mmp14 and Nos2 in mono- or coculture microglia.

Analysis of the expression of different genes in microglia cells transfected with the indicated siRNA cultured in medium alone or segregated coculture for 6 hours with GL261 glioma cells. Results are presented relative to those of Ctrl siRNA transfected BV2 cells cultured alone, set as 1. Statistics and error bars: mean ± s.e.m. n=6 of biological replicates. Data were analyzed using two-tailed Student’s t-test. *P< 0.05; **P< 0.01; ***P< 0.001; ****P< 0.0001.

Supplementary Figure 4 Analysis of the expression of Casp3 and Casp8 in BV2 microglial cells after segregated coculture with C6 cells.

(a,b) Microglial mRNA expression level for Casp3 (a) and Casp8 (b) upon 6 (black bars) and 24h (white bars) segregated coculture with C6 glioma cells. Results are presented relative to those of BV2 microglia cultured in medium alone, set as 1. Statistics and error bars: mean ± s.d. n=3 of biological replicates. Data was analyzed using two-tailed Student’s t-test. ns, not significant (P > 0.05);*P< 0.05; **P< 0.01.

Supplementary Figure 5 Role of thioredoxins in the S-nitrosylation of caspase-3 after 6 h of segregated coculture with C6 cells.

(a) Immunoblots showing knockdowns of Trx1 (top) and Trx2 (bottom) expression in BV2 microglia cells using different siRNA pools. (b) Effect of PX-12 (9µM), a specific Trx1 inhibitor, over microglial DEVDase activity upon glioma segregated coculture.(c) Immunoblots representing the levels of total Trx2 (top) and SNO-Trx2 (bottom) in BV2 cells upon segregated coculture with C6 cells using the biotin-switch method (results represented at the bottom of the panel). (d,e) Analysis of Trx1 and Trx2 (d) and Trx Reductase 1 and Trx Reductase 2 (e) activities in cytosolic (black bars) and mitochondrial (white bars) fractions in BV2 microglia cells after segregated coculture with C6 cells for 6h. Results in each of the panels are presented relative to those of BV2 cells cultured alone, set as 1. Statistics and error bars: mean ± s.d. except d,e which shows mean ± s.e.m. n=4 (b,d,e) and n= 3 (c) of biological replicates. Data was analyzed using two-tailed Student’s t-test. ns, not significant (P > 0.05);*P< 0.05 and **P< 0.01.

Supplementary Figure 6 Analysis of microglial cell density 6 months after tamoxifen treatment in Casp3flox/floxCx3cr1CreERT2 mice.

(a) Analysis of microglia caspase-3 expression 6 months after tamoxifen treatment in Casp3flox/floxCx3cr1CreERT2 mice to induce the specific deletion of Casp3 gene in microglia cells. Casp3flox/flox mice were used as control. Tamoxifen was administered in 7 day-old-mice following standard procedures. The purity of the microglia preparation was evaluated by flow cytometry analysis using CD11b and CD45 antibodies. Genomic DNA was isolated and deletion of the Casp3 floxed sequence was evaluated by qPCR analysis following an ABC primer strategy. Microglia from Casp3flox/floxCx3cr1CreERT2 mice had 63 % Casp3 gene deletion 6 months after inducing the deletion. Results are presented relative to Casp3flox/flox mice, set as 100. (b) This panel shows a representative illustration of Iba1-labeled microglia in the striatum of Casp3flox/floxCx3cr1CreERT2 and Casp3flox/flox mice 6 months after tamoxifen treatment. (c,d) Quantification of the effect of Casp3 gene deletion on microglia cell population in striatum and cortex. Statistics and error bars: mean ± s.d. n=3. Data were analyzed using two-tailed Student’s t-test. ns, not significant (P > 0.05); ****P< 0.0001. Scale bar= 200 μm.

Supplementary Figure 7 Proposed pathway.

(a) Schematic representation of the suggested pathway of how glioma cells induce microglial caspase-3 S-nitrosylation, decreasing caspase-3 proteolytic activity and promoting their conversion towards a tumor-supporting activation state. (b) Illustration of how caspase-3 like (DEVDase) activity based on its degree of activation will regulate different microglial activation states (tumor-supporting versus pro-inflammatory phenotypes), or in some circumstances with very high activity levels, even cell death.

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Shen, X., Burguillos, M., Osman, A. et al. Glioma-induced inhibition of caspase-3 in microglia promotes a tumor-supportive phenotype. Nat Immunol 17, 1282–1290 (2016). https://doi.org/10.1038/ni.3545

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