CSF1R inhibition depletes tumor-associated macrophages and attenuates tumor progression in a mouse sonic Hedgehog-Medulloblastoma model

Abstract

The immune microenvironment of tumors can play a critical role in promoting or inhibiting tumor progression depending on the context. We present evidence that tumor-associated macrophages/microglia (TAMs) can promote tumor progression in the sonic hedgehog subgroup of medulloblastoma (SHH-MB). By combining longitudinal manganese-enhanced magnetic resonance imaging (MEMRI) and immune profiling of a sporadic mouse model of SHH-MB, we found the density of TAMs is higher in the ~50% of tumors that progress to lethal disease. Furthermore, reducing regulatory T cells or eliminating B and T cells in Rag1 mutants does not alter SHH-MB tumor progression. As TAMs are a dominant immune component in tumors and are normally dependent on colony-stimulating factor 1 receptor (CSF1R), we treated mice with a CSF1R inhibitor, PLX5622. Significantly, PLX5622 reduces a subset of TAMs, prolongs mouse survival, and reduces the volume of most tumors within 4 weeks of treatment. Moreover, concomitant with a reduction in TAMs the percentage of infiltrating cytotoxic T cells is increased, indicating a change in the tumor environment. Our studies in an immunocompetent preclinical mouse model demonstrate TAMs can have a functional role in promoting SHH-MB progression. Thus, CSF1R inhibition could have therapeutic potential for a subset of SHH-MB patients.

Figure 1.. Increased number of TAMs is associated with tumor progression in a sporadic SHH-MB mouse model.

(A) Schematic representation of experimental design. (B) Principal component analysis (PCA) of progressing (P; n=4) and regressing tumors (R; n=3), which defined as having a >50% increase or >20% decrease in size between 7–11 weeks, respectively. (C) 3D MEMRI volume renderings of the brain of progressing (green) or regressing (blue) tumors in Atoh1-SmoM2 mice. (D) Tumor progression curves of progressing and regressing tumors. (E) Percentage of CD45⁺CD11B⁺ myeloid cells among all immune cells in 25 Atoh1-SmoM2 tumors (n=15 mice). (F) Number of CD45⁺CD11B⁺ myeloid cells per milligram (mg) of tumor in progressing and regressing tumors. (G–N) Number of indicated cell types per mg of tumor. Data are pooled from 3 FACS experiments. Mean ± SD. Significance was determined using unpaired t test, ****p<0.0001, **p<0.01, *p<0.05.

Figure 2.. The amount of immune cell infiltration in human SHH-MB samples does not correlate with driver mutation.

(A–D) Representative immunohistochemical staining of Ki67 (A), CD3 (B), CD68 (C) and CD163 (D) from 20 SHH-MB samples. (E–H) Correlation between the staining of Ki67 (E), CD3 (F), CD68 (G) and CD163 (H) and driver mutation. Quantification was done on the region that had the strongest positive signal and a score was given to each sample based on the expression and distribution of positive cells as indicated in (I–J). Mean ± SD. Significance was determined using one-way ANOVA with Tukey post hoc pairwise comparison.

Figure 3.. Depletion of Tregs or T cells in Atoh1-SmoM2 mice does not alter mouse survival.

(A) Kaplan–Meier curves of Atoh1-SmoM2 mice treated with IgG control antibody or anti-CD25 antibody (PC61) antibody starting at 3 weeks. (B) Schematic representation of Treg depletion experimental design. (C) Tumor volume of mice after given 3 doses of PC61or IgG control antibody (8 weeks of treatment). (D–F) Flow cytometry analysis of control (n=3 mice, 5 tumors) and PC61-treated (n=4 mice, 7 tumors) showing percentage of Tregs among CD4⁺ T cells (C), percentage of CD8⁺ T cells among TCRb⁺ T cells (D) and percentage of CD45⁺ CD11b⁺ myeloid cells among CD45⁺ immune cells (E). (G) Kaplan–Meier curves of Atoh1-SmoM2-Rag1 heterozygous null (control) and Atoh1-SmoM2-Rag1 homozygous null (no mature B and T cells) littermates. m.s. = median survival. Significance was determined using Log-rank test for (A, G) and unpaired t test for (C–F). **p<0.01, ns = non-significant.

Figure 4.. Depletion of TAMs prolongs mouse survival and blocks tumor progression in most Atoh1-SmoM2 mice.

(A) Kaplan–Meier curves of control (CTRL)- or PLX5622 (PLX)-treated Atoh1-SmoM2 mice. m.s. = median survival. (B) Schematic representation of experimental design. (C) 3D MEMRI volume renderings of the brain (grey) and tumors (magenta) in CTRL and PLX-treated mice at the indicated time points. (D) The overall change in tumor volume between the pre-treatment time point (V_initial; 7 weeks) and the experimental endpoint (V_final; mice became symptomatic or at 13 weeks). Black line indicates median. (E–F) Waterfall plots showing the change in tumor volume from the pre-treatment time to after 2 weeks (E) and 4 weeks (F) of CTRL or PLX treatment. (G) The growth rate calculated as the percentage tumor volume change per day. The open circles indicate outlier PLX tumors that grew faster than the controls. Mean ± SD. Significance was determined using Log-rank test for (A) and Mann-Whitney test for (D–G). **p<0.01, *p<0.05, ns = non-significant.

Figure 5.. Atoh1-SmoM2 tumors treated for 4 weeks with PLX show a decrease of monocytes/macrophages and an increase of CD8 cytotoxic T cells.

(A) Experimental design. (B) Change in tumor volume between pre-treatment and 4 weeks of PLX treatment. The open circle in (B, E–G, I–K) indicates an outlier PLX tumor. Black line indicates median. (C) Flow cytometry quantification of myeloid cell composition in CTRL (n=12) and PLX (n=14) treated tumors. Significance was found in the CD45^int TAM and microglia-like TAMs populations (See Sup Fig. 10). (D–G) Representative flow cytometry panels from the CD45⁺CD11B⁺Ly6G⁻ population (D) showing the proportion of CD45^int TAMs (E) and microglia-like TAMs (F). (G) Percentage of CD45^hi TAMs of mixed myeloid population. (H–J) Representative flow cytometry panel from the lymphocytes (CD45⁺CD19⁺TCRβ⁺) showing the proportion of CD8⁺ T cells (I), CD4 T⁺ cells (J). (K) Percentage of B cells (CD45⁺CD19⁺B220⁺) of the lymphocyte population. Data are pooled from 3 FACS experiments. Mean ± SD. Significance was determined using Mann-Whitney test for (B), and performed excluding the outlier tumor by using unpaired t test for (C), (E–G) and (I–K), ***p<0.001, **p<0.01, *p<0.05.

Figure 6.. Depletion of TAMs in Atoh1-SmoM2 mice leads to an increase of pro-inflammatory T cells.

(A) Schematic representation of experimental design. (B–D) Number of CD8⁺ T cells (B), CD4⁺ T cells (C), and CD45⁺ immune cells (D) per milligram (mg) of tumor in control (CTRL)- and PLX-treated tumors. (E) Percentage of Granzyme B⁺ (Gzmb) CD107⁺CD8⁺ T cells of total CD8⁺ T cells. (F) Number of Gzmb⁺ CD107⁺CD8⁺ T cells per mg of tumor. (G–I) Representative flow cytometry panels from the CD45^hi TCRβ⁺ CD8⁺ population (G) showing the proportion (H) and the number (I) of IFNγ⁺CD8⁺ T cells. (J–L) Representative flow cytometry panels from the CD45^hiTCRβ⁺CD4⁺ population (J) showing the proportion (K) and the number (L) of IFNγ⁺CD4⁺ T cells. Mean ± SD. Significance was determined using unpaired t test for (B–F), (H–I) and (K–L). **p<0.01.

Figure 7.. Depletion of TAMs in Atoh1-SmoM2 mice leads to a pro-inflammatory tumor microenvironment.

(A–F) Analysis of MHC II expression in 12 CTRL tumors and 14 PLX tumors (see Fig. 5). The open circle indicates an outlier PLX tumor. Representative flow cytometry panels from the CD45^int TAMs population (A) showing the proportion of MHC II⁺ CD45^int TAMs (B). Representative flow cytometry panels from the microglia-like TAMs population (C) showing the proportion of MHC II⁺ microglia-like TAMs (D). Representative flow cytometry panels from the CD45^hi TAMs population (E) showing the proportion of MHC II⁺ CD45^hi TAMs (F). Mean ± SD. Significance was performed excluding the outlier tumor by using unpaired t test. **p<0.01.