Immunotherapies against brain metastases have shown clinical benefits when applied to asymptomatic patients, but they are largely ineffective in symptomatic cases for unknown reasons. Here, we dissect the heterogeneity in metastasis-associated astrocytes using single-cell RNA sequencing and report a population that blocks the antitumoral activity of infiltrating T cells. This protumoral activity is mediated by the secretion of tissue inhibitor of metalloproteinase-1 (TIMP1) from a cluster of pSTAT3+ astrocytes that acts on CD63+ CD8+ T cells to modulate their function. Using genetic and pharmacologic approaches in mouse and human brain metastasis models, we demonstrate that combining immune checkpoint blockade antibodies with the inhibition of astrocyte-mediated local immunosuppression may benefit patients with symptomatic brain metastases. We further reveal that the presence of tissue inhibitor of metalloproteinase-1 in liquid biopsies provides a biomarker to select patients for this combined immunotherapy. Overall, our findings demonstrate an unexpected immunomodulatory role for astrocytes in brain metastases with clinical implications.

Significance:

This study presents a significant advancement in understanding immune modulation in brain tumors and offers new insights into the potential therapeutic interventions for brain metastases.

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©2024 The Authors; Published by the American Association for Cancer Research
2024
American Association for Cancer Research
This open access article is distributed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0) license.
This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License .
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Supplementary data

List of supplementary figure, supplementary tables and authors included in the RENACER signature

Supplementary Data file containing the list of supplementary figure legends (S1-S9) and their equivalence to the corresponding main figure and supplementary tables (Supplementary Table 1-25) with a short description as well as the authors included in the RENACER signature

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Table S1

Astrocyte signatures. Signatures used for astrocytes annotation in the single-cell RNA sequencing of the brain metastasis-associated microenvironment using the B16/F10-BrM model. The table contains information corresponding to Figure 1B and Supplementary Figure 1C.

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Table S2

DEG cluster 3 brain metastasis-associated astrocytes. Differentially expressed genes (DEGs) found in cluster number 3 of brain metastasis associated-astrocytes. Significantly deregulated genes are highlighted. Red indicates upregulation and green indicates downregulation respect to tumor free condition. The table contains information corresponding to Figure 1D.

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Table S3

DEG cluster 7 brain metastasis-associated astrocytes. Differentially expressed genes (DEGs) found in cluster number 7 of brain metastasis associated-astrocytes. Significantly deregulated genes are highlighted. Red indicates upregulation and green indicates downregulation respect to wt condition. The table contains information corresponding to Figure 1E.

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Table S4

GSEA cluster 3, cluster 6 and cluster 7 of brain metastasis-associated astrocytes. Gene set enrichment analysis (GSEA) of top 25 upregulated and downregulated signatures comparing cluster number 3/6/7 of brain metastasis associated-astrocytes with the tumor free condition. The table contains information corresponding to Figure 1D-E and Supplementary Figure 1G-H.

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Table S5

Analysis of potential interactions cluster 3-cluster 7. Top 25 interactions of potential secreted and potential membrane proteins in cluster number 3 and cluster number 7 of brain metastasis associated-astrocytes considering the experimentally determined interaction. The table contains information corresponding to Supplementary Figure 1M.

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Table S6

Human brain metastasis samples analyzed with Chromium Fixed RNA Profiling. Clinical information of human brain metastasis samples included in Figure 1F-I.

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Table S7

STAT3-activation pathways in brain metastasis-associated astrocytes in human brain metastasis samples. Gene set enrichment analysis (GSEA) of STAT3-activation pathways in STAT3+ human brain metastasis associated-astrocytes. No significant alterations were found in cluster 3. The table contains information corresponding to Supplementary Figure 1P.

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Table S8

GSEA_comparision between cluster 3 and cluster 5 of brain metastasis-associated astrocytes in human brain metastasis samples. Gene set enrichment analysis (GSEA) of upregulated and downregulated signatures comparing cluster number 3 and number 5 of human brain metastasis associated-astrocytes. The table contains information corresponding to Figure 1I.

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Table S9

GSEA_comparision between cluster 4 and cluster 5 of brain metastasis-associated astrocytes in human brain metastasis samples. Gene set enrichment analysis (GSEA) of upregulated and downregulated signatures comparing cluster number 4 and number 5 of human brain metastasis associated-astrocytes. The table contains information corresponding to Figure 1I.

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Table S10

GSEA_comparision between STAT3+ clusters of brain metastasis-associated astrocytes in human brain metastasis samples. Gene set enrichment analysis (GSEA) of upregulated and downregulated signatures comparing each STAT3+ cluster and the other clusters of brain metastasis-associated astrocytes. The table contains information corresponding to Figure 1F-I.

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Table S11

GSEA of CD8+ T cells incubated with pSTAT3+ conditioned media. Gene set enrichment analysis (GSEA) of top 25 upregulated and downregulated signatures and NES of Biological Process (GOBP) pathways related to T cell function comparing CD8+ in vitro cultures incubated with the pSTAT3- (d1) or pSTAT3+ (d2) secretome. The table contains information corresponding to Figure 2B.

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Table S12

RENACER immune cluster evaluation. Immune cluster and gene expression of immune markers in the RENACER cohort of human brain metastasis samples. The table contains information corresponding to Supplementary Figure 4C-D.

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Table S13

Human brain metastases evaluated with multiplex. Information regarding multiplex of immune cells and quantification of TIMP1 RNAScope staining in human brain metastasis samples. The table contains information corresponding to Figure 3G.

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Table S14

Human brain metastases refractory to immune checkpoint blockade. Clinical information of samples from brain metastasis patients that responded to immune checkpoint blockade systemically but not in the brain. The table contains information corresponding to Figure 3H.

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Table S15

Patient-derived organotypic cultures (PDOC) treated with anti-TIMP1. Results from the Patient-derived organotypic brain cultures (PDOCs) of human brain metastasis treated with Anti-TIMP1 or Anti-TIMP1 and CD8 blockade. The table contains information corresponding to Figure 4F-G.

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Table S16

Predictive analysis of patient-derived organotypic cultures (PDOC) treated with anti-TIMP1. Clinical information of samples treated in patient-derived organotypic brain cultures (PDOCs) of human brain metastasis with Anti-TIMP1 stratified accorded to their Immune Cluster. The table contains information corresponding to Supplementary Figure 5M-N.

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Table S17

GSEA analysis of proteomic data obtained from CD8+ T cells incubated with astrospheres conditioned medium. NES and statistics of the comparisons between the following experimental conditions: CD8+ lymphocytes were cultured with pSTAT3- astrospheres conditioned medium (CM) (D1) and wt (D2) or cKOGFAP-Timp1 (D2KO) pSTAT3+ astrospheres CM processed for phosphoproteomic analysis. The table contains information corresponding to Supplementary Figure 7B.

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Table S18

Phosphoproteomic analysis of CD8+ T cells incubated with astrospheres conditioned medium. Results from phosphopeptides quantification of the LC-MS/MS analysis of CD8+ lymphocytes incubated with pSTAT3- (D1) and wt (D2) or cKOGFAP-Timp1 (D2KO) pSTAT3+ astrospheres conditioned medium. The table contains information corresponding to Figure 6K and Supplementary Figure 7A.

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Table S19

Human brain metastases evaluated for pERK1/2 regarding their immune cluster. Information regarding multiplex of immune cells in human brain metastasis samples used for assessing pERK1/2 status in CD8+ T cells. The table contains information corresponding to Figure 6M.

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Table S20

Statistics of multiple experimental arms from in vivo immunotherapy experiments. Unpaired two-tailed Student’s t-test of different comparisons between the following experimental conditions: IgG2, silibinin and immune checkpoint blockade (ICB) (Anti-PD1 plus Anti-CTLA4) alone or in combination with silibinin in mice intracardially injected with B16/F10-BrM cells. Unpaired two-tailed Student’s t-test of different comparisons between the following experimental conditions: IgG2, cKOGFAP-Timp1 and immune checkpoint blockade (ICB) (Anti-PD1 plus Anti-CTLA4) alone or in combination in mice intracardially injected with E0771-BrM cells. The table contains information corresponding to Figure 7C and Supplementary Figure 8L.

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Table S21

TIMP1 levels from ELISA applied to CSF from patient samples. Levels of TIMP1 in the blood or in the cerebrospinal fluid (CSF) of non-cancer patients and brain metastasis patients from different primary tumors. Immune Cluster is shown for patients in Figure 7N. The table contains information corresponding to Figure 7L, 7N and Supplementary Figure 9A,C-D.

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Table S22

Overall survival according to TIMP1 in the CSF. Overall survival of brain metastasis patients with different primary tumors according to their levels of TIMP1 in the cerebrospinal fluid (CSF). The table contains information corresponding to Supplementary Figure 9B.

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Table S23

Antibodies used for immunohistochemistry. Detailed information of antibodies used for immunohistochemistry stainings.

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Table S24

Antibodies used for multiplex immunohistochemistry. Detailed information of antibodies used for multiplex.

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Table S25

Antibodies used for flow cytometry analysis. Detailed information of antibodies used for flow cytometry.

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Figure S1

Heterogeneity within the brain metastasis microenvironment. Supplementary Figure 1 shows additional data corresponding to Figure 1.

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Figure S2

STAT3-dependent immunosuppression in brain metastasis. Supplementary Figure 2 shows additional data corresponding to Figure 2.

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Figure S3

TIMP1 is a downstream target of STAT3 in brain metastasis-associated astrocytes. Supplementary Figure 3 shows additional data corresponding to Figure 3.

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Figure S4

Local immunosuppression could explain immunotherapy failure in high immune cluster symptomatic brain metastasis. Supplementary Figure 4 shows additional data corresponding to Figure 3.

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Figure S5

TIMP1 mediates brain metastasis in a CD8+ T cell-dependent manner. Supplementary Figure 5 shows additional data corresponding to Figure 4.

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Figure S6

TIMP1 through CD63 alters relevant properties of CD8+ T cells. Supplementary Figure 6 shows additional data corresponding to Figure 6.

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Figure S7

TIMP1 binding to CD63 modulates kinase signaling in CD8+ T cells. Supplementary Figure 7 shows additional data corresponding to Figure 6.

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Figure S8

A combined immunotherapy targeting local immunosuppression provides superior control of brain metastasis. Supplementary Figure 8 shows additional data corresponding to Figure 7.

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Figure S9

Patient-derived organotypic cultures as a surrogate to assess the potential of targeting local immunosuppression in brain metastasis. Supplementary Figure 9 shows additional data corresponding to Figure 7.

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