Remote neuronal activity drives glioma progression through SEMA4F

Abstract

The tumour microenvironment plays an essential role in malignancy, and neurons have emerged as a key component of the tumour microenvironment that promotes tumourigenesis across a host of cancers^1,2. Recent studies on glioblastoma (GBM) highlight bidirectional signalling between tumours and neurons that propagates a vicious cycle of proliferation, synaptic integration and brain hyperactivity^3-8; however, the identity of neuronal subtypes and tumour subpopulations driving this phenomenon is incompletely understood. Here we show that callosal projection neurons located in the hemisphere contralateral to primary GBM tumours promote progression and widespread infiltration. Using this platform to examine GBM infiltration, we identified an activity-dependent infiltrating population present at the leading edge of mouse and human tumours that is enriched for axon guidance genes. High-throughput, in vivo screening of these genes identified SEMA4F as a key regulator of tumourigenesis and activity-dependent progression. Furthermore, SEMA4F promotes the activity-dependent infiltrating population and propagates bidirectional signalling with neurons by remodelling tumour-adjacent synapses towards brain network hyperactivity. Collectively our studies demonstrate that subsets of neurons in locations remote to primary GBM promote malignant progression, and also show new mechanisms of glioma progression that are regulated by neuronal activity.

Extended Data Figure 1.. Contralateral activity drives glioma progression and infiltration

a. Kaplan-Meier survival analysis of RCAS-Ntva tumors treated with saline (median_Saline= 95 days, n = 11) or CNO (median_CNO = 51 days, n = 9) showing significantly faster morbidity in CNO treated RCAS tumors (Log-rank (Mantel-Cox) test, Chisq = 6.456, df = 1, p-value = 0.0111, CNO/Saline HR_log-rank= 2.768, 95% CI = 0.9770 to 7.945). b. H&E staining of RCAS-Ntva tumors samples revealed high grade characteristics in CNO treated tumor groups (red arrows). Ki67 staining proliferation in CNO treated mice versus saline treated mice. Quantification is derived from n=5 mice from CNO (mean = 14.51%, SD = 7.076%) and Saline (mean = 4.017%, SD = 2.179%) groups and determined by Welch’s unpaired t-test (P-value = 0.0276, t = 3.228, df = 4.441). c. Mathematical modeling of glioma infiltration as a function of tumor mass. Blue line is the smoothed data points using piecewise-cubic splines; red horizontal dashed lines are the 0.8 p_max and 0.02 p_max glioma cell density of the maximum smoothed cellular density (p_max ). Red vertical lines are the intersecting distance points of the red horizontal lines with smoothed blue line, which is used in calculating infiltrating width (IW). Black arrow shows the IW. Log-log plot shows the dependence of IW and tumor mass (TM). Analysis was performed at the p30 timepoint on CTL n = 3, Saline n = 3, CNO n = 3; samples from individual biological replicates are color coded. d. Glioma 3D spheroid migration assay, measuring glioma infiltration after treatment with growth factor media, conditioned media (CM) from spontaneously active cortical explants, spontaneously active cortical explants silenced with TTX (10μm) or optogenetically stimulated cortical explants (channelrhodopsin-2 (ChR2)-expressing deep layer cortical projection neurons), in comparison to ACSF control. Data are plotted as median (center line), IQR (box limits) and minimum and maximum values (whiskers) (b).

Extended Data Figure 2.. Contralateral cortical stimulation accelerates tumor progression

a. Schematic depicting injection of AAV into the contralateral cortex. b. Electrophysiology measuring neural activity in response to CNO treatment on mouse brain sections to confirm DREADD activity. c. Schematic of intra-uterine electroporation (IUE) model of high-grade glioma (HGG). Representative images of tumor brains from 20 day old mice versus 60 days old. Tumor brain slices were stained with Hoechst, and native GFP fluorescence was utilized to visualize tumor. Time course of contralateral cortical infiltration. Log-regression of 3xCr tumors demonstrated that infiltration correlates strongly with time (two-sided log-regression, Chi-square = 23.38, df = 1, p-value <0.0001, n₃₀ =22, n₅₀=19, n₇₀=21, n₉₀=10). 72 IUE-HGG bearing mice were generated from 10 separate litters of mice and sampled across P30, P50, P70, P90 to ensure accurate representation of infiltration. d. Representative images of infiltrating tumor across the midline at each respective timepoint. e. Representative Ki67 staining of CNO-treated versus Saline-treated versus untreated IUE-HGG sections taken from P30 tumor brains. f. Representative Ki67 staining of CNO-only control, without hM3Dq, Saline, and IUE-HGG sections taken from P30 tumor brains, g-h. Representative images of infiltrating tumor across the midline at each respective timepoint from the CNO-only control experiments (g) and saline only control (h). Tumor sections were imaged and quantified for each condition from at least five mice to ensure reproducibility (d-h).

Extended Data Figure 3.. Ipsilateral stimulation does not promote early tumor progression

a. Representative Ki67 staining of CNO versus Saline versus untreated IUE-HGG sections taken from P30 tumor brains after ipsilateral stimulation. b. Representative images of corpus callosum cut tumors stained for Ki67 (quantification in main figure). Tumor samples were prepared as described above, using native GFP to image tumor. c. Representative images of Rasgrf2-dCre tumors stained for Ki67 (quantification in main figure). Tumor samples were prepared as described above, using native GFP to image tumor). Tumor sections were imaged and quantified for each condition from at least five mice to ensure reproducibility (a-c).

Extended Data Figure 4.. Activation of contralateral inhibitory neurons does not promote tumor progression

a. Representative image of Rosa-LSL-TdTom + Rasgrf2-dCre mouse brain harvested 2 days post Trimethoprim treatment. Cells from layer 2/3 of the cortex show TdTom labelling. b. Representative images from IUE-HGG tumors at P30 demonstrating infiltration and Ki67 expression after activation of inhibitory neurons with AAV-Dlx5/6-hM3Dq in the cortex contralateral. c. Quantification of tumor infiltration and Ki67 expression at P30. Infiltration analyzed one-way ANOVA. Data derived from 3xCr CTL (n=7), 3xCr + DLX-hM3Dq-mCh + Saline (n=6), 3xCr + DLX-hM3Dq-mCh + CNO (n=7) tumors with 18 coronal slices analyzed per tumor. Coronal sections with infiltrating tumor showed no difference in proliferation compared to saline treated (p-value = 0.1823) or control tumors (p-value = 0.5328) or infiltration at P30 compared to Saline (p-value = 0.8880) or control tumors (p-value = 0.9981). d. Stacked bar chart of SingleR labeled populations percent representation in CNO and Saline single cell sequencing datasets. e. Feature plot of cluster of interest marker genes. Color represents a score assigned based on overall expression of marker genes. Seurat’s AddModuleScore function was used to calculate these scores. Red Arrow denotes population of interest featured in Fig. 3B. f. GO terms of genes enriched in the spatial transcriptomics analysis of the leading edge of P50 mouse tumors (R6-R8), (Cutoff for DEG via DESeq2, with p-value < 0.05). Data are plotted as median (center line), IQR (box limits) and minimum and maximum values (whiskers) (c).

Extended Data Figure 5.. Expression characteristics of EphA6, EphA7, and Sema4F

a. Heatmap of IVY-GAP expression data of all axon guidance genes included in the bar coded screen described in main figure 4a. b. Heatmap of IVY-GAP expression data for Sema4f, EphA6, EphA7. c. KM curve of GEPIA survival data. GBM patient cohort data were split based on high and low Sema4F expression. d. Western blots of wild type, GOF, and LOF tumors. (pBSema4F is GOF; sgSema4F is LOF). Antibodies and concentrations used are described in methods. Western blots were quantified in ImageJ. Western blots were performed on biological replicates (2 additional tumor sample) to ensure reproducibility.

Extended Data Figure 6.. Analysis of EphA6, EphA7, and Sema4F GOF/LOF tumors

a. Kaplan-Meier survival curve of individual gain-of-function and loss-of-function validation studies for EphA6 and EphA7. EphA6-GOF (median_A6GOF = 105 days, Chisq = 0.7, df=1, p-value =0.4, n=11), EphA6-LOF (median_A6LOF = 133days, Chis =7.2, df=1, p-value=0.007, n = 7), EphA7-GOF (median_A7GOF= 97 days, Chisq=0, df=1 p-value=0.9, n=15), EphA7-LOF (median_A7LOF= 97.5 days, Chisq=1.3, df=1, p-value=0.3). b. Representative immunostainings of EphA6, EphA7, and Sema4F human tumor micro-array. c. Quantification of infiltration from these tumors across the P30-P70 timecourse. Infiltration was quantified based on the presence of tumor cells in contralateral cortex and analyzed via two-way analysis of variance (ANOVA). Error bars represent standard deviation, data derived from EphA6-GOF p30 n=7, EphA6-GOF p50 n=13, EphA6-GOF p70 n=11, EphA6 CTL p30 n=8, EphA6 CTL p50 n=6, EphA6 CTL p70 n=11, EphA6-LOF p30 n=8, EphA6-LOF p50 n=6, EphA6-LOF p70 n=11, EphA7-GOF p30 n=9, EphA7-GOF p50 n=12, EphA7-GOF p70 n=12, EphA7 CTL p30 n=5, EphA7 CTL p50 n=5, EphA7 CTL p70 n=7, EphA7-LOF p30 n=6, EphA7-LOF p50 n=6, EphA7-LOF p70 n=9. d. Quantification of transwell migration of human glioma cell lines; infiltrating cells were counted after 48 hours incubation (n=3 wells per condition). e. Kaplan-Meier survival curve for human glioma cell lines transplanted into mouse brain. Samples were analyzed via log-rank (Mantel-Cox) test. WT median survival=66 days, n=11; GFP median=62 days n=7; Sema4f-GOF median=74 days, n=10, Chi = 0.4120, p-value = 0.5209; shSCR median=69 days, n=9; shSema4F median = undefined after 100 days, n=8, Chi = 14.08 p-value = 0.0002. *P < 0.05, **P < 0.01, ***P < 0.001, **** P < 0.0001, two-way analysis of variance (ANOVA) (c), one-way analysis of variance (ANOVA) (f), Log-rank (Mantel-Cox) test (a,d). Data are plotted as median (center line), IQR (box limits) and minimum and maximum values (whiskers) (c-d).

Extended Data Figure 7.. Infiltrating glioma from EphA6 and EphA7 GOF/LOF tumors

a. Representative images of IUE-HGG GOF/LOF/CTL of EphA6 in P50 mice. Frozen sections were stained with Hoechst and native GFP was assessed for infiltration status into the contralateral cortex. b. Representative images of IUE-HGG GOF/LOF/CTL of Epha7 in P50 mice. Samples assessed as outlined above (EphA6-GOF p50 n=13, EphA6 CTL p50 n=6, EphA6-LOF p50 n=6, EphA7-GOF p50 n=12, EphA7 CTL p50 n=5, EphA7-LOF p50 n=6) (a,b). c. Representative H&E staining of IUE-HGG tumors containing GOF and LOF of EphA6, EphA7, and Sema 4F. All tumors demonstrated high-grade characteristics such as microvascular proliferation or necrosis regardless of GOF or LOF. H&E staining repeated on n=4 tumor samples per condition d. qRT-PCR validation of Sema4F-GOF (S4F-GOF) overexpression and shRNAi knockdown in human glioma cell lines. Data are derived from n=3 biological replicates per condition. Results are plotted as mean +/− SD. Values were analyzed by Brown-Forsythe and Welch two-way ANOVA. S4F-GOF showed significant upregulation compared to uninfected control (**P=0.0082). shS4F #4 showed significant downregulation compared to shScramble cells (*P=0.0167).

Extended Data Figure 8.. Representative Ki67 images from Sema4F manipulated tumors

a. Representative images of Sema4F GOF/LOF and control tumors stained for Ki67 in P30 mice. Tumor samples were using native GFP to image tumor. b. Representative images of Sema4F-LOF and control tumors treated with CNO or Saline and stained for Ki67 in P30 mice. c. Representative images from IUE-HGG tumors at P30 demonstrating the extent of infiltration with Sema4F-ectodomain and Sema4F-ectodomain + Sema4F-LOF. d. Quantification of tumor infiltration and Ki67 expression at P30. Infiltration analyzed one-way ANOVA. Data derived from n=6 CTL, n=7 S4F-Ecto, and n=5 S4F Ecto+LOF tumors, *P=0.0483, **P_infil=0.0025, **P_Ki67=0.0023 ****P=<0.0001. e. Representative images of Sema4F-ectodomain and Sema4F-ectodomain + Sema4F-LOF and control tumors stained for Ki67 in 30 day old mice. f. Representative images of infiltrating tumor across the midline from Sema4Fectodomain and Sema4F-ectodomain + Sema4F-LOF tumors in 30 day old mice. Data are plotted as median (center line), IQR (box limits) and minimum and maximum values (whiskers) (d).

Extended Data Figure 9.. Single Cell RNA-Seq analysis

a. Single Cell RNA-Seq DimPlots of P50 IUE-HGG from CNO, Sema4F, and saline controls UMAP plot of full single cell datasets from CNO and Saline treated and Sema4F GOF tumors. Tumors were isolated and sequenced as outlined above. All clusters were represented in all datasets, though their relative abundance varied. b. Single cell RNASeq analysis of from IUE-glioma model, sub-clustering on non-tumor, GFP-negative cells. Red circle denotes neuronal populations, marked by Map2, Tubb3, and Ncam. Note that Plexin B1 and B2 expression is enriched in neuronal populations, denoted by purple dots. c. Volcano plot depicting the differentially expressed genes (DEGs) between human glioma cell lines overexpressing Sema4F (Sema4F-GOF) and controls. For downregulated genes with blue color: padj<0.001 & log2FoldChange<(-0.75); for upregulated genes in red: padj<0.001 & log2FoldChange>0.75) d. GO analysis of DEGs upregulated in the Sema4F-GOF glioma cell lines. DEGs from padj<0.001 & log2FoldChange>0.75)

Extended Data Figure 10.. Staining for synaptic markers in mice bearing PDXSema4F-GOF tumors

a. Antibody staining of inhibitory synapses (VGAT-Gephryin) and b. excitatory (Vglut2PSD95) synapse from P50 mouse brains at peritumoral margins from PDX tumors ovexpressing Sema4F and control. Box denotes zoomed in region in adjacent panel (20X, 63X, and 200X magnification left to right; scale bar left to right: 50 um, 20 um and 10 um). Quantification of synaptic staining via unpaired two-tailed t-test is derived from PDX-Con n=3 and PDX-S4F-GOF n=3, utilizing a total of 13 fields of view from these sections for each experimental condition, (***P=0.0001, ****P<0.0001), data are presented as mean values +/− SE.

Figure 1.. Remote neuronal stimulation accelerates glioma progression

a. Schematic of DREADD-based activation of neurons contralateral to IUE tumor. b. Representative images from IUE-HGG tumors at P30 after contralateral activation or control; green is tumor, red is AAV-DREADD virus. c. Quantification of infiltration and Ki67 expression via two-way ANOVA analysis. Analysis of infiltration derived from P30 CTL n = 8, P50 CTL n = 7, P70 CTL n = 7, P30+CNO n = 8, P50+CNO n = 7, P70+CNO n = 7, P30+Saline n = 8, P50+Saline n = 7, P70+Saline n = 7, P30+AAV+Saline n = 8, P50+AAV+Saline n = 5, P30+AAV+Saline n = 4, P30+AAV+CNO n = 8, P50+AAV+CNO n = 6, P30+AAV+CNO n = 5 samples. (***P=0.0001, *P=0.0107). Ki67 staining was performed at the P30 time point, from CTL n = 9, CNO-only n = 8, Saline n = 7, AAV+Saline n = 5, AAV+CNO n = 4 samples (****P<0.0001). d. Representative images from IUE-HGG tumors at P30 after ipsilateral neuronal activation; green is tumor, red is AAV-DREADD virus. e. Quantification of infiltration and Ki67 expression at P30 via two-way ANOVA. Analysis is derived from 3xCr CTL n = 6, 3xCr+IpsilAAV+Saline n=7, 3xCr+IpsilAAV+CNO n=9. CNO stimulated brains revealed no statistical difference with Saline treated (P=0.1247) or control tumors (P=0.2714). Comparison between ipsilateral-CNO and contralateral-CNO groups revealed a statistically significant difference (****P=0.0001). Ki67 staining was performed on 3xCr CTL n=5, 3xCr+IpsilAAV+Saline n=5, 3xCr+IpsilAAV+CNO n=5 (****P<0.0001, ***P=0.0003); Data are plotted as median (center line), IQR (box limits) and minimum and maximum values (whiskers) (c, e). Scale bars are 1000um.

Figure 2.. Callosal projection neurons promote glioma progression

a. Representative images from IUE-HGG tumors at P30 demonstrating infiltration with a severed corpus callosum (cut-CC) or control (no cut); green is tumor; red is AAV-DREADD virus. b. Quantification of infiltration and Ki67 expression at P30 via two-way ANOVA. Analysis is derived from AAV+CNO (No Cut) n = 5, AAV+Saline (No Cut), n = 4, AAV+CNO (Cut) n = 6, CC-cut+AAV+Saline (Cut) n = 6 (****P<0.0001). c-d. Schematic of callosal projection neuron activation experiment and Rasgrf2-dCre mouse line with Cre-inducible DIO-hM3D-2a-mCherry DREADD to selectively activate layer2/3 neurons in contralateral hemisphere. e. Representative images from IUE-HGG tumors, injected with AAV-DIO-hM3D-2a-mCherry in Rasgrf2-Cre mice; green is tumor, red is AAV-DIO-DREADD virus. f. Quantification of infiltration and Ki67 expression at P30 via one-way ANOVA. Analysis is derived from Rasgrf2+AAV+CNO n=9, Rasgrf2+AAV+Saline n=6, 3xCr+AAV+CNO n=6, 3xCr+AAV+Saline n=6. (*P=0.0225, ***P=0.0015, **P=0.011). Data are plotted as median (center line), IQR (box limits) and minimum and maximum values (whiskers) (b,f). Scale bars are 1000um.

Figure 3.. Identification of activity-dependent infiltrating glioma population

a. Single Cell RNA-Seq DimPlots of P50 IUE-HGG from CNO and saline controls. b. Sub-clustering analysis of GFP+ tumor cells shown in a; red arrow indicates the cluster of interest. GO-term analysis on upregulated (average log FC>0, P<0.05) genes was performed using EnrichR and the KEGG 2021 Human dataset. c. Spatial transcriptomics on P50 IUE-HGG from the CNO group; R1-R2 denote ipsilateral tumor, R3-R5 denote tumor within corpus callosum, R6-R8 denote infiltrating tumor in contralateral hemisphere. d. Heatmap depicting the expression of markers associated with the single cell cluster of interest from b and their relative expression across the tumor regions displayed in c. Fold change of markers from the scRNA-Seq data are mapped in pink and blue. e. Heatmap depicting the enrichment of markers associated with the single cell cluster of interest and their relative expression across various anatomical locations in human GBM, derived from the IVY-GAP database. These enrichment scores were generated with AUCell analysis (depicted in red and blue), or ssGSEA analysis (depicted in yellow and blue) and plotted as a heatmap

Figure 4.. In vivo screen identifies Sema4F as a driver of glioma progression

a. Schematic of barcoded screen, tumors were harvested from ipsilateral primary and contralateral secondary tumor sites. b. Next-generation sequencing for barcode amplification. Samples were derived from n=4 biological replicates with paired primary tumor, secondary tumor, and input library samples to derive fold change relative to starting abundance. Results are plotted as mean +/− SEM. c. Kaplan-Meier survival curve of individual gain-of-function and loss-of-function for Sema4F. Sema4F-GOF (median_S4FGOF=83 days, Chisq = 14.8, df = 1 p-value = 0.0001, n = 44), Sema-LOF (median_S4FLOF=112 days, Chisq = 3.9, df = 1 p-value = 0.05, n = 10), controls (n=18) d. Representative images from IUE-HGG tumors at P50 from Sema4F-GOF, Sema4F-LOF, or control groups demonstrating infiltration; green is tumor. e. Quantification of infiltration from these tumors across the P30-P70 time course via two-way ANOVA. Analysis is derived from Sema4F-GOF p30 n=14, Sema4F-GOF p50 n=14, Sema4F-GOF p70 n=10, Sema4F CTL p30 n=7, Sema4F CTL p50 n=6 Sema4F CTL p70 n=7, Sema4F-LOF p30 n=17, Sema4F-LOF p50 n=16, Sema4F-LOF p70 n=19. (*P=0.0462, **P=0.001, ****P<0.0001). f. Representative images from IUE-HGG tumors at P30 after combined Sema4F-LOF and neuronal activation (CNO); green is tumor, red is AAV-DREADD virus. g. Quantification of tumor infiltration and Ki67 expression at P30 analyzed via two-way ANOVA. Analysis is derived from Sema4F-KO+AAV+Saline n=7, Sema4F-KO+AAV+CNO n=8, 3xCr CTL n=5, 3xCr+AAV+Saline n=5, 3xCr+AAV+CNO n=6. (*P=0.0195, ***P=0.0003, ****P=<0.0001). Scale bars are 1000um.

Figure 5.. Sema4F promotes synaptic remodeling and brain hyperactivity

a. Single Cell RNA-Seq DimPlots of P50 IUE-HGG from Sema4F-GOF, CNO, and saline controls; shown is the GFP+ tumor cluster. Infiltrating tumor subpopulation is highlighted in green and denoted by red arrow. b. Sub-clustering analysis of tumor cells. Analysis of altered expression in this unique prospective cell population (in green) was performed using EnrichR and the KEGG 2021 Human dataset. c. Antibody staining of excitatory (Vglut2-PSD95) and inhibitory synapses (VGAT-Gephryin) P50 mouse brains at peritumoral margins from Sema4F-GOF and control tumors; box denotes zoomed in region in adjacent panel (10X and 200X magnification left to right; white scale bar is 12.5μm and yellow scale bar is 200μm). d. Quantification of synaptic staining. Images were sampled from n=3 biological replicates, with n=13 CTL, n=15 S4F GOF, n=16 S4F LOF fields sampled for Vglut/PSD95 colocalization, and n=23 CTL, n=19 S4F GOF, n=16 S4F LOF fields sampled for Vgat/Gephryn colocalization. Results presented as mean +/− SD and ****P<0.0001). e. Sample EEG traces from mice bearing control or Sema4F-GOF tumors. f. Quantification of spikes/hr over a 24-hr period at one-week intervals from P50 to P64. Spikes were recorded in 3 mice per condition and analyzed via two-way ANOVA (*P=0.024) and plotted as mean values +/− SD . Data are plotted as median (center line), IQR (box limits) and minimum and maximum values (whiskers) (c,f).