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. 2021 May 15;13(10):2400.
doi: 10.3390/cancers13102400.

Targeting CSF1R Alone or in Combination with PD1 in Experimental Glioma

Justyna M Przystal  1   2 , Hannes Becker  1   2 , Denis Canjuga  1 , Foteini Tsiami  1   2 , Nicole Anderle  3 , Anna-Lena Keller  3 , Anja Pohl  3 , Carola H Ries  4 , Martina Schmittnaegel  4 , Nataliya Korinetska  1 , Marilin Koch  1 , Jens Schittenhelm  2   5 , Marcos Tatagiba  1   6 , Christian Schmees  3 , Susanne C Beck  1   2 , Ghazaleh Tabatabai  1   2   7
Affiliations

Targeting CSF1R Alone or in Combination with PD1 in Experimental Glioma

Justyna M Przystal et al. Cancers (Basel). .

Abstract

Glioblastoma is an aggressive primary tumor of the central nervous system. Targeting the immunosuppressive glioblastoma-associated microenvironment is an interesting therapeutic approach. Tumor-associated macrophages represent an abundant population of tumor-infiltrating host cells with tumor-promoting features. The colony stimulating factor-1/ colony stimulating factor-1 receptor (CSF-1/CSF1R) axis plays an important role for macrophage differentiation and survival. We thus aimed at investigating the antiglioma activity of CSF1R inhibition alone or in combination with blockade of programmed death (PD) 1. We investigated combination treatments of anti-CSF1R alone or in combination with anti-PD1 antibodies in an orthotopic syngeneic glioma mouse model, evaluated post-treatment effects and assessed treatment-induced cytotoxicity in a coculture model of patient-derived microtumors (PDM) and autologous tumor-infiltrating lymphocytes (TILs) ex vivo. Anti-CSF1R monotherapy increased the latency until the onset of neurological symptoms. Combinations of anti-CSF1R and anti-PD1 antibodies led to longterm survivors in vivo. Furthermore, we observed treatment-induced cytotoxicity of combined anti-CSF1R and anti-PD1 treatment in the PDM/TILs cocultures ex vivo. Our results identify CSF1R as a promising therapeutic target for glioblastoma, potentially in combination with PD1 inhibition.

Keywords: CSF1R; PD1; glioblastoma; immunotherapy; sequential therapy.

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Conflict of interest statement

J.M.P., H.B., D.C., N.K., J.S., M.T., and S.C.B. declare no conflicts of interest. M.K. received the Scholarship for Interdisciplinary Oncology including accommodation costs from medac GmbH as well as travel and accommodation costs from Roche for neuro-oncology training. C.H.R. is a former Roche employee and an inventor on granted and pending patent applications for therapeutic CSF1R antibodies. M.S. is a current member of Roche Diagnostics GmbH. G.T. reports personal fees from B.M.S., AbbVie, Novocure, Medac, and Bayer, and grants from B.M.S., Novocure, Roche Diagnostics, and Medac outside the submitted work. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Figures

Figure 1
Figure 1
CSF1R and PD1 are present in primary and progressive glioblastoma. Representative tumor areas from matched pairs of newly diagnosed and progressive glioblastoma. H&E staining (top row) and immunohistochemical staining of CSF1R (n = 28), CD204 (n = 27), CD163 (n = 31), PD1 (n = 30), PD-L1 (n = 31), CD3 (n = 28), CD4 (n = 30), and CD8 (n = 28). Scale bars 50 µm.
Figure 2
Figure 2
Monotherapies with PD1 and CSF1R blockade in experimental syngeneic SMA-560 glioma in vivo. (A): Kaplan–Meier plot showing symptom-free survival. Experimental groups (n = 10 in each group) include control treatment (saline), anti-CSF1R (2G2) antibody, anti-PD1 (RPM1.14) antibody, and respective control antibodies. Treatments started on day 14 post-tumor implantation. Tukey–Kramer post hoc test was used after performing Log-rank (Mantel–Cox) test (p < 0.001). ** p < 0.001 Survival time depicted in Kaplan–Meier plot refers to experimental endpoint as described in detail in material/methods section and in Supplementary Table S1. (B,C): Immunohistochemical analysis in post-treatment SMA-560 gliomas of one representative animal per group (n = 1). Small inserts show staining control without application of primary antibody. (scale bars in (B): 100 μm; scale bars in (C): 50 μm).
Figure 3
Figure 3
Simultaneous and sequential combinations of PD1 and CSF1R blockade in vivo. (A): Schematic overview of experimental design. (B): Kaplan–Meier plots: combination therapies vs. monotherapies vs. controls. Experimental groups (n = 10 in each group) are as indicated: Blue dashed line shows p-value between combination therapy group starting with anti-CSF1R treatment and CSF1R control group. Green dashed lines show p-value between both control groups vs. simultaneous combination group. P-values were calculated by using Tukey–Kramer post hoc test after performing Log-rank (Mantel–Cox) test (p < 0.0001). (C): Symptom-free survival graph displaying each single mouse per experimental group. Experimental groups are: control anti-PD1, control anti-CSF1R, anti-PD1, anti-CSF1R, anti-CSF1R plus anti-PD1, anti-CSF1R and then anti-PD1, and anti-PD1 and then 2nd anti-CSF1R. Dashed line on day 16 represents median latency until experimental endpoint in control group. Dashed line on day 17 shows time point where last animal of control group reached experimental endpoint. Day 52 indicates last surviving animals. Experimental endpoints are described in detail in the material/methods section and in Supplementary Table S1.
Figure 4
Figure 4
Immunohistochemical analysis in post-treatment tissues (n = 3 in each group were analysed). Representative IHC staining patterns of tumor tissues with indicated antibodies after 2 injections of CSF1R antibodies and 3 injections of PD1 antibodies. Small inserts show staining control without application of primary antibody. Scale bars 50 µm.
Figure 5
Figure 5
Quantification of Ki67, cleaved caspase 3, CD4, CD8 as well as CD8/CD4, and CD8/FoxP3 ratios in post-treatment tissue. Quantification of Ki67 (A), cleaved caspase 3 (B), CD4 (C), CD8+ (D), CD8+/CD4+ ratio (E), FoxP3+ (F), CD8/FoxP3 ratio (G), CD163 (H), and CD204 (I) in tumor tissues after 2 injections of CSF1R and 3 injections of PD1 antibodies. Three animals (n = 3) in each group were analysed. Statistical analysis was done using one-way ANOVA followed by Tukey’s multiple comparison test. ** p < 0.01, * p < 0.05.
Figure 6
Figure 6
Immunohistochemical analysis of one representative animal per group (n = 1) of tumor-infiltrating host cells in simultaneous versus sequential combinations of PD1 and CSF1R blockade in vivo. (A), H&E and immunohistochemical analysis in representative tumor tissues. Scale bar 100 μm. (B), Immunohistochemical analysis in representative tumor tissues. Scale bars 50 μm. Small inserts show staining control without application of primary antibody. (C), Quantification of CD8+ (1), CD8+/CD4+ ratio (2), and CD204+ (3) cells. For quantification, three tissue samples of different tumor depth per animal were analysed. Statistical analysis was done using one-way ANOVA followed by Tukey’s multiple comparison test. p < 0.05.
Figure 7
Figure 7
Treatment-induced cytotoxicity in PDMs and PDM/TILs coculture. (A) Immunohistochemistry staining of (1) PDM model 1 and (2) PDM model 2 for markers of macrophages (CD68), tumor-associated macrophages markers (CD204 and CD163), and CSF1R. Scale bars 100 µm. (B) PDM model 1, coculture with autologous TILs, treatments and concentrations as indicated after 72 h (n = 3 per concentration). Fold changes were normalized to PDMs only. Two-way ANOVA followed by Dunnett’s multiple comparison test was used. PDMs+IgG4-Control served as control group. **** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05. (C): PDM model 2 was treated in the absence of TILs with either CSF1R/ PD1 or combination treatments and concentrations as indicated. Cytotoxicity was measured after 72 h. Fold changes were normalized to isotype control; significance above bars refer to control group. Two-way ANOVA followed by Dunnett’s multiple comparison test was used. PDMs +IgG4-Control served as control group. *** p < 0.001, * p < 0.05.
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