Abstract The subcellular distribution of lysosomes, the main degradative organelles of mammalian cells, responds to metabolic cues in a highly dynamic way. While lysosomal positioning due to amino acid levels is well-characterized, cholesterol-dependent regulation of lysosomal motility is incompletely understood. We explored impaired lysosomal cholesterol export using a mass spectrometry-based multi-OMICs approach, identifying widespread reallocation of resources and signaling pathway modulation. We identified increased phosphorylation at LAMTOR1 serine 56 in response to cholesterol level perturbations. We demonstrate that this phosphorylation site is sufficient to disrupt Rag GTPases/SLC38A9 binding to the Ragulator complex, inhibiting canonical mTORC1 and facilitating binding of BORC, therefore promoting lysosomal retrograde movement. LAMTOR1 S56 phosphorylation responds exclusively to depletion of lysosomal limiting membrane cholesterol, is facilitated by mTOR, and presents a negative feedback loop for amino acid independent displacement of Ragulator bound Rag GTPases, limiting canonical mTORC1 activity. Mass spectrometry data are available via ProteomeXchange with identifier PXD073489. Highlights Perturbation of lysosomal cholesterol homeostasis results in adaptation of cellular protein and lipid biosynthesis LAMTOR1 is phosphorylated at serine 56 via mTORC1 LAMTOR1 S56 phosphorylation is lysosomal membrane cholesterol dependent LAMTOR1 S56 phosphorylation disrupts binding of Rag GTPases to the Ragulator complex LAMTOR1 S56 phosphorylation promotes binding of Ragulator to BORC, facilitating lysosomal retrograde transport

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

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.

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

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.

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

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.

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.

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.

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.

The autophagy-lysosomal pathway is crucial for maintaining homeostasis and survival of neurons, hence defects in this system have been associated with neurodegeneration, including Parkinson's disease (PD). The cysteine proteases cathepsin B (CTSB) and cathepsin L (CTSL) are involved in the clearance of various neurodegenerative disease-related proteins such as amyloid- , huntingtin and the prion protein. While there are studies implicating CTSB and CTSL as mediators of α-synuclein/SNCA clearance, their exact roles remain unclear. We previously demonstrated that recombinant procathepsin D can enhance the clearance of pathological-aggregates of SNCA both in vitro and in vivo, as well as restoring autophagy function. These results prompted us to investigate the role of the two cysteine proteases CTSB and CTSL regarding SNCA degradation by dosing recombinant human procathepsin B (rHsCTSB) and procathepsin L (rHsCTSL) alone or in combination. We here demonstrate that both proteases are efficiently endocytosed by neuronal cells and transported to lysosomes, where they undergo maturation into active enzymes. Treatment with either rHsCTSB or rHsCTSL resulted in a reduction of different SNCA species, present in Triton-insoluble protein fractions as well as sensitive for various pathology- and structure-specific antibodies analyzed via Western blot, immunofluorescence and ELISA. These effects were found to be similar in all models used here: dopaminergic neurons derived from induced pluripotent stem cells (iPSC) of PD patients harboring the SNCA A53T mutation, ex vivo organotypic brain slices and primary neuronal cultures of human SNCA overexpressing Thy1 mice. Interestingly, our data so far do not indicate a synergistic effect of both cysteine cathepsins when applied together. As proof-of-concept for future therapeutic studies, intracranial injections of both recombinant enzymes reduced SNCA in brains of a transgenic mouse model (Ctsd knockout) harboring SNCA pathology. Moreover, treatment with recombinant CTSB and CTSL improved lysosomal/autophagy functions indicated by recovery of β-glucocerebrosidase (GCase) activity and SQSTM1 (p62) level. Further, SNCA-dependent synaptic defects as well as toxicity was reduced after treatment of neuronal cells. These findings suggest that enhancing lysosomal CTSB or CTSL effectively degrades pathology-associated SNCA, suggesting a potential therapeutic protease-based strategy for PD and other synucleinopathies.

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

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

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.

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.

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.

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.See related commentary by Lorger and James, p. 11

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.

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.

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