Extended Data Fig. 5: Clozapine does not affect the tumour immune microenvironment, and ORN activity affects glioma progression.

(a) Proliferation rates of two CKO model-derived glioma cell lines treated with clozapine as indicated. The cells were maintained in complete medium. The proliferation rates of culture cells were measured by EdU incorporation assay (n = 4 biologically independent samples for each group). (b–d) Quantification of tumour invasiveness (c) and tumour proliferation (d) in the CKO model under clozapine treatment at the tumour stage. Tumour invasiveness was defined as the distance from the core tumour area (with the highest density in the tumour) to the normal area (where the density of tumour cells declined to the same as that in the region away from the tumour), as the length of white rectangular (b). Five random areas were calculated for each mouse (n = 5 mice for each group). The tumour mice were from the same group of mice used in the first two columns of Fig. 2d. (e) Percentage of granulocytes, macrophage, dendritic cells, B lymphocytes and T lymphocytes in the peripheral blood of the WT mice (at the age of 8 weeks) under clozapine treatment. Clozapine injection, clozapine was administered by i.p. (two injections per week for three weeks). Clozapine drinking, mice were access to clozapine containing water for three weeks (n = 4 mice for each group). For FACS gating strategy, see Supplementary Fig. 2. (f) Percentage of granulocytes, macrophage, dendritic cells, B lymphocytes and T lymphocytes in the peripheral blood of the WT, Omp-hM3Dq and Omp-hM4Di transgenic mice (at the age of 8 weeks) under clozapine treatment. The clozapine treatment was the same as that of injection paradigm in (e). Clozapine was administered by i.p. (two injections per week for three weeks). w/ clozapine, with clozapine. The values are presented as the ratio to the WT mice (n = 4 mice for each group). (g) The blood-brain barrier integrity was evaluated by Evans Blue dye. w/o Evans Blue, without Evans Blue treatment. w/ Evans Blue, with Evans Blue treatment. WT mice at the age of 8 weeks were used. The schemes of clozapine treatment by drinking water or i.p. injection are the same as that in (e). See Methods section for the details (n = 4 mice for each group). (h) Immunochemistry staining of CD15 in the brain of WT, Omp-hM3Dq, Omp-hM4Di and the CKO tumour mice with clozapine treatment. Spleen was used as the positive control. Red arrows point to CD15⁺ granulocytes in the spleen. WT mice treated with vehicle was used as negative control. T, tumour area. The brain samples of the WT, Omp-hM3Dq, Omp-hM4Di mice were from the mice analysed in (f). The brain samples of CKO tumour mice were from the same group of clozapine-treated CKO mice used in Fig. 2d. (i) Quantification of Iba1⁺ cell density in CKO_Omp-hM4Di model at the pre-transforming stage as indicated in Extended Data Fig. 4j (n = 6 OBs for CKO, n = 6 OBs for CKO with clozapine drinking, n = 4 OBs for CKO_Omp-hM4Di, n = 6 OBs for CKO_Omp-hM4Di with clozapine drinking). P value: column A vs B, 0.9932; A vs C, 0.3648; A vs D, 0.9984; B vs C, 0.4936; B vs D, 0.9728; C vs D, 0.2964. (j) Quantification of Iba1⁺ cell density in CKO_Omp-hM3Dq model at the pre-transforming stage as indicated in Extended Data Fig. 4l (n = 6 OBs for CKO with vehicle, n = 6 OBs for CKO with clozapine, n = 4 OBs for CKO_Omp-hM3Dq with vehicle, n = 6 OBs for CKO_Omp-hM3Dq with clozapine). P value: column A vs B, 0.7722; A vs C, 0.6589; A vs D, 0.8465; B vs C, 0.2175; B vs D, 0.3158; C vs D, 0.9725. (k–m) Quantification the density of total macrophage/microglia (marked by Iba1⁺) (k) and those activated macrophage/microglia (CD68⁺Iba1⁺) (l) in the non-tumour region (k, l) or the tumour region (m, n) of the CKO tumour brain. Non-tumour region is defined as the brain region where no obvious tumour lesion was included (usually far away from the tumour core). Clozapine was administered by drinking water from P30 to P210. Mice were analysed at P210 (n = 6 OBs for CKO and n = 4 OBs for CKO with clozapine drinking in (k). n = 4 OBs for each group in (l–n)). (o) Representative images of the macrophage/microglia of the CKO model analysed in (k–n). Low-power and high-power images were both provided. The MERGE channel in low-power images including DAPI, tdT and Iba1 channels. The MERGE channel in high-power images including DAPI, CD68, tdT and Iba1 channels. N, non-tumour region. T, tumour region. Of note, the samples analysed in (k–o) were from the same group of CKO mice used in Fig.2d (the first two columns). (p) Representative images of mTq2 and tdT expression in glomeruli of CKO_Omp-hM4Di mice administered with clozapine. (q and r) Quantification of mTq2 (assumed also hM4Di) and tdTomato intensity among glomeruli of CKO_Omp-hM4Di mice with (q) and without (r) clozapine treatment. Each dot represents one glomerulus. Two-sided Pearson correlation coefficients were calculated. (s and t) Immunofluorescence staining (s) and Western blots (t) of tyrosine hydroxylase (TH) in control and left-naris occlusion mice. Mice were plugged at P31 and analysed after two months. For gel source data, see Supplementary Fig. 1. (u–w) Scheme of the experimental procedure (u) and for single naris occlusion analysis at the pre-transforming stage. Proliferation cell density (v) and proliferation rate of mutant cells (w) were analysed (n = 4 mice for each group). (x–z) Scheme of the experimental procedure (x), representative whole brain images (y) and quantification (z) of L-naris occlusion mice grafted with mouse glioma cell lines. Scale bars: (b), 100 μm; (o), 500 μm and 50 μm; (p), 400 μm; (s), 1000 μm. One-way ANOVA with Dunnett’s multiple comparison post-hoc test in (a), (e), (f), (g), (i) and (j). One-sided t-test in (c), (d), (k), (l), (m), (n), (v), (w) and (z). NS, not significant, ****p < 0.0001. Data are mean ± s.e.m. The box plot marks the median, upper and lower quartiles and whiskers are presented min to max in (v) and (w). See also Methods section for details of each part of the experiment.

Source Data