Immune cell infiltration into brain tumor microenvironment is mediated by Rab27-regulated vascular wall integrity

Abstract

Aggressive brain tumors often exhibit immunologically 'cold' microenvironment, where the vascular barrier impedes effective immunotherapy in poorly understood ways. Tumor vasculature also plays a pivotal role in immunoregulation and antitumor immunity. Here, we show that small GTPase Rab27 controls the vascular morphogenesis and permeability for blood content and immune effectors. Thus, in Rab27a/b double knock out (Rab27-dKO) mice, the brain vasculature is abnormally scarce, while the blood vessels become dysmorphic and hyperpermeable in the context of brain tumors, including syngeneic glioblastoma. These defects are reflected in rearrangements of endothelial cell subpopulations with underlying diminution of venous endothelial subtype along with changes in gene and protein expression. Notably, Rab27-dKO brain endothelial cells exhibit deficient tight junctions, whereby they enable large-scale extravasation of cytotoxic T cells into the tumor mass. We show that Rab27-regulated vascular T cell infiltration can be exploited to enhance adoptive T cell therapy in syngeneic brain tumors.

Fig. 1.. Rab27 controls vascular morphogenesis in the brain.

(A) Immunostaining of primary BECs isolated from Rab27-WT mice for CD31 (red), Rab27a (green), and Rab27b (cyan), with 4′,6-diamidino-2-phenylindole (DAPI) (blue) counterstain, imaged at 63× using super-resolution microscopy (SIM). (B) Western blot normalized quantifications to study levels of Rab27a and Rab27b in WT (white), dHET (gray), and dKO (blue) primary BEC isolates. (C to E) Immunostaining of the vasculature using CD31 (red) and DAPI (blue) counterstain on brain tissues derived from Rab27-WT (C), Rab27-dHET (D), and Rab27-dKO (E) mice. (F) Cartoon to show murine neocortex (top) and quantification of total microvascular density in the neocortex (bottom) of WT (white), dHET (gray), and dKO (blue) mice. (G) Layer-related microvascular density counts in the neocortex of WT (white), dHET (gray), and dKO (blue) mice. (H to M) Tube formation assay using primary WT-BECs treated with DMSO (H), 1 μM Nex-20 (I), 2.7 μg of IgG (J), 2.7 μg of anti-Rab27b antibody (K), DMSO + IgG (L), and 1 μM Nex-20 + 2.7 μg of anti-Rab27b antibody (M). (N to Q) Quantifications of the number of nodes (N), junctions (O), meshes (P), and branches (Q), formed by primary WT-BECs after 15 hours in culture. WT, wild type (C57bl/6); dHET, Rab27a/b double heterozygote; dKO, Rab27a/b double knockout; *P < 0.05 and **P < 0.01. Scale bars, 100 µm.

Fig. 2.. Rab27 deficiency leads to dysmorphic vasculature in murine GBM.

(A) Schematic of experimental design. (B) Kaplan-Meyer survival curve of WT (black), dHET (gray), and dKO (blue) mice injected intracranially with GL261 murine glioma cells and monitored for survival. n = 5 in each group. (C to E) Immunostaining of brain glioma tissues for CD31 in WT (C), dHET (D), and dKO (E). (F) Quantification of microvascular density for brain tumor tissues of WT (white), dHET (gray), and dKO (blue) GL261 tumor models. (G) Quantification of tumor blood vessel size/diameter in WT (white), dHET (gray), or dKO (blue) mice bearing GL261 intracranial lesions. WT, wild type (C57bl/6); dHET, Rab27a/b double heterozygote; dKO, Rab27a/b double knockout; T, tumor; L, vascular lumen; not significant (ns); *P < 0.05 and ****P < 0.0001.

Fig. 3.. Rab27 deficiency leads to abnormal transcriptional phenotypes of EC subpopulations.

(A) UMAP plot for scRNAseq for BECs and TECs in mice with either dHET or dKO genotype. (B) UMAP plot for the different cell-type clusters previously reported (49). (C) Bar graph to compare the percentage of genes observed across the different cell-type clusters in BECs and TECs. (D) Dot plot to compare the five most differentially regulated genes in the different subsets of ECs. The differentially regulated genes are listed on the y axis, and the different EC-type clusters for either dHET (green) or dKO (pink) are on the x axis. The red font on labels for the x axis indicates the tumor-derived groups of ECs for dHET or dKO, respectively. The size of each dot is based on the percent of gene expression as is shown in the key. BECs, primary BECs; TECs, primary tumor-derived BECs; aEC, arterial ECs; cEC, capillary ECs; cEC_arterial, capillary ECs close to the arterial branches; cEC_venous, capillary ECs close to the veins; vEC, vein ECs; aEC_large, large artery ECs; aEC_stress, arterial ECs with features of stress response; cEC_angiogenic, angiogenic capillary ECs; cEC_venous_stress, capillary ECs with venous-like phenotype and features of stress response; vEC_activated, activated venous ECs; vEC_TM_survival, venous ECs specific for the tumor microenvironment enriched for markers of cell survival; vEC_TM_IFN, venous ECs with features reflective of tumor microenvironment enriched for markers of inflammation; vEC_TM, venous ECs with tumor-associated characteristics; vEC_TM_prolif, venous ECs with features specific for the tumor microenvironment enriched in proliferation markers.

Fig. 4.. In Rab27-deficient mice, tumor blood vessels lose junctional structures.

(A) Schematic of BEC isolation by FACS using anti-CD31⁺ antibody in preparation for mass spectrometry. (B) Left: Venn diagram to show the number of identified EC proteins in BEC isolates from either dHET or dKO mice. Protein threshold of 99.0%, peptide threshold of 95%, and minimum of 1 peptide in Scaffold program. Label-free quantitation was conducted by normalized total spectra in Scaffold. Right: Volcano plot to show the substantially affected proteins in the proteomes of dHET- or dKO-derived BECs. (C) Left: Venn diagram to show the number of identified endothelial proteins in TEC isolates of dHET and dKO mice harboring GL261 brain tumors. Protein threshold of 99.0%, peptide threshold of 95%, and minimum of 1 peptide in Scaffold program. Label-free quantitation is conducted by normalized total spectra in Scaffold. Right: Volcano plot to show the substantially affected proteins in TECs isolated from either dHET or dKO tumor bearing mice. (D to F) Validation of selected proteins down-regulated in the dKO-BEC proteome relative to dHET-BEC proteome. Immunofluorescence staining for CD31, ZO-1, phalloidin, and DAPI counterstain in untreated Rab27-WT–derived BECs (D), Rab27-WT–derived BECs treated with either IgG + DMSO (E) or with 1 μM Nex20 + (1:100) anti-Rab27b antibody (F). (G and H) GL261 brain tumor tissue sections stained for CD31 and ZO-1. Tissues were isolated from tumors in dHET (G) or dKO (H) mice. (I) Quantification of colocalization between CD31 and ZO-1 in GL261 brain tumors. (J and K) GL261 brain tumor tissue sections stained for CD31 and Cldn5, following tumor formation in either dHET (J) or dKO (K) mice. (L) Quantification of colocalization between CD31 and Cldn5 brain tumor bearing mice. Images were procured using the super-resolution microscope at 63× and processed by structured illumination (SIM) calculation, followed by maximum intensity projection to combine the z stacks; ****P < 0.0001.

Fig. 5.. Disruption of Rab27 compromises vascular barrier function in brain tumor microenvironment.

(A) Entry into brain parenchymal of Evans bue/Hoechst dye cocktail as a function of differential vascular permeability in WT (top), dHET (middle), and dKO (bottom) mice harboring GL261 intracranial tumors. (B) Quantification of the intensity of Evans blue tissue containment as measured using ImageJ package. (C) Quantification of the intensity of Hoechst, as measured by ImageJ. (D) Quantification of CD8α⁺ cell infiltration into brain tumors in WT, dHET, or dKO mice. (E) Schematic of the experimental design for testing vascular permeability and T cell extravasation into GL261 intracranial tumors. (F) Visualization of blood vessels (lectin, blue), T cells (anti-CD8α–PE, red), and cancer cells (GL261-GFP, green) in tumors of dHET (top) and dKO (bottom) mice using 250-μm-thick vibratome sections. (G) Quantification of CD8α⁺ cells located intravascularly (top-left) and in the extravascular tumor parenchyma (top-right) and the total number of CD8α⁺ cells in both compartments (bottom) counted manually; dHET mice (gray); dKO mice (blue). BECs, primary BECs; dHET, Rab27a/b double heterozygote; dKO, Rab27 double knockout. Images were generated using confocal microscopy at 63× and processed through maximum intensity projection to combine the z stacks; *P < 0.05, **P < 0.01, and ****P < 0.0001.

Fig. 6.. Impact of targeting TJ machinery (Cldn5) in ECs resembles the consequences of Rab27 deficiency.

(A) Schematic representation of in vivo experiment to study effects of Cldn5 inhibitor, M01 on the brain tumor vasculature. (B) M01 safety profile; weights of C57bl/6 mice during the course of M01 administrations indicate no systemic toxicity in the animals exposed to the adopted treatment regimen. (C) Kaplan-Meier curves documenting the absence of notable prosurvival effects of M01 alone (versus PBS) in GL261 brain tumor bearing mice. (D) Immunostaining for CD31 in sections of GL261 brain tumors following treatment with M01 or PBS demonstrates the emergence of larger vessel sizes in the M01 group. (E and F) Quantification of microvascular density (E) and vessel diameter (F) in GL261 brain tumor–bearing mice treated with PBS (white) or M01 (blue). (G) Schematic representation of M01 administration scheme before subjecting the mice to magnetic resonance imaging (MRI) for vascular permeability assessment. (H) Representative MRI scans of the brains of mice treated with PBS (top) and M01 (bottom) with GADOVIST as a contrast agent. (I) Quantification of the different regions of the brains after M01 administration shows leaky vasculature in the M01 group relative to controls. (J) Immunohistochemistry for CD8α in brain tumor tissues of animals treated with PBS or M01. (K) Quantification of CD8α numbers per tumor area in animals treated with PBS or M01. *P < 0.05 and **P < 0.01.

Fig. 7.. Rab27 inhibition enables adoptive T cell immunotherapy of mouse brain tumors.

(A) Schematic depicting the experimental design involving CFSE-labeled, OT-1 T cells and their infiltration across the EC barrier in vitro. (B) BECs treated with Nex-20 (1 μM), anti-Rab27b antibody (27 μg/ml), and Rab27b blocking peptide (25 μg/ml) mimic the consequences of Rab27 deficiency. (C) Schematic of adoptive T cell therapy experiments. (D) CFSE-labeled effector OT-1 T cell infiltration into GL261-OVA tumor microenvironment in mice with indicated genotypes; top: experimental design; bottom: the content of CFSE⁺ T cells migrating into brain tumors of dKO and dHET mice as measured by FACS. (E) Long-term effects of effector OT-1 T cell systemic adoptive immunotherapy in dHET or dKO mice baring GL261 or GL261-OVA brain tumors. OT-1 T cell therapy had no impact on GL261 tumors but prolonged survival of mice with GL261-OVA tumors in both dHET and dKO mice. The effect of OT1 T cell systemic therapy in dKO mice harboring GL261-OVA significantly increased mouse survival relative to dHET counterparts. BECs, primary BECs; dHET, Rab27a/b double heterozygote; dKO, Rab27 double knockout; RFU, relative fluorescence units. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.